Methods and compositions for regulation of cell death in plants

ABSTRACT

The present invention comprises a novel, positive regulator of cell death in Arabidopsis. This gene, LOL1, plays a role in the positive regulation of cell death in both an lsd1 mutant poised to undergo runaway cell death and in wild type plants challenged with pathogens. In another aspect of the invention, LOL1 provides enhanced disease resistance.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is based on and claims priority to U.S. ProvisionalApplication Ser. No. 60/326,534, filed Sep. 28, 2001, hereinincorporated by reference in its entirety.

TECHNICAL FIELD

The present invention generally relates to plant breeding and plantgenetics. More particularly, the invention relates to a novel positiveregulator of programmed cell death and disease resistance. The inventionalso relates to plants and plant lines in exhibiting disease resistanceand those in which the novel positive regulator of programmed cell deathis disposed. Methods of breeding and engineering such plants and plantlines are also disclosed. PCD programmed cell death HR hypersensitiveresponse ROI reactive oxygen intermediates SA salicylic acid MAP mitogenactivated protein RCD runaway cell death T_(m) melting point TMV tobaccomosaic virus MCMV maize chlorotic mottle virus AMV alfalfa mosaic virus

Single-Letter Three-Letter Code Code Name A Ala Alanine V Val Valine LLeu Leucine I Ile Isoleucine P Pro Proline F Phe Phenylalanine W TrpTryptophan M Met Methionine G Gly Glycine S Ser Serine T Thr Threonine CCys Cysteine Y Tyr Tyrosine N Asn Asparagine Q Gln Glutamine D AspAspartic Acid E Glu Glutamic Acid K Lys Lysine R Arg Arginine H HisHistidine

Functionally Equivalent Codons

Amino Acid Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGUAspartic Acid Asp D GAC GAU Glumatic acid Glu E GAA GAG PhenylalaninePhe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAUIsoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU Leucine Leu L UUA UUG CUACUC CUG CUU Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S ACG AGUUCA UCC UCG UCU

BACKGROUND ART

Plant biology is replete with examples of programmed cell death (PCD),yet very little is known about the relevant control mechanisms. The mostfamiliar form of plant PCD is the hypersensitive response (HR)associated with successful plant innate immune responses (Dangl et al.,in Biochemistry and Molecular Biology of Plants (Buchanan et al., eds.)(ASPP Press, Rockville, 2000) pp. 1044-1100; Morel & Danal, (1997) CellDeath and Different. 19: 17-24; Shirasu & Schulze-Lefert, (2000) PlantMolec. Biol. 44: 371-385). Here, recognition of a pathogen leads torapid ion fluxes, production of reactive oxygen intermediates (ROI) andnitric oxide, MAP kinase signaling, transcriptional re-programming inand around the infection site, salicylic acid (SA) biosynthesis, andcell collapse (Danal & Jones, (2001) Nature 411: 826-833; Fevs & Parker,(2000) Trends Genet. 16: 449-455; McDowell & Danul, (2000) TrendsBiochem. Sci. 25: 79-82). HR is required for disease resistance in someplant-pathogen interactions (Peterhänsel et al., (1997) Plant Cell 9:1397-1409), but could simply reflect the passing of a signal thresholdleading to cell death in others (Bendahmane et al., (1999) Plant Cell11: 781-791).

Several loss of function mutations define cell death control genes inArabidopsis (Dietrich et al., (1994) Cell 77: 565-578; Greenbera &Ausubel, (1993) Plant J. 4: 327-342; Walbot et al., in GeneticEngineering of Plants, (Kosuge & Meredith, eds.) Plenum Publishing Co.,New York, 1983, vol. 3, pp. 431-442). These mutations typically alsoinduce disease resistance responses, suggesting that they either aretrue negative regulators of HR cell death or that the ectopic cell deaththey engender is sufficient to activate defense pathways. The genes theyencode are not related, to date, to commonly defined metazoan cell deathregulators (Jones & Danal, (1996) Trends Plant Science 1:114-119;Korsmeyer, (1995) Trends Genet. 11: 101-105).

Two null mutant phenotypes suggest that the Arabidopsis LSD1 gene is animportant negative regulator of plant PCD (Dietrich et al., (1997) Cell88: 685-694). First, lsd1 plants are unable to control cell deathinitiated by pathogen recognition or by signaling chemicals that mimicSA action (Dietrich et al., (1994) Cell 77: 565-578). lsd1 is thuspoised for a runaway cell death (RCD) phenotype. It has beendemonstrated that superoxide is necessary and sufficient to control thispoise (Jabs et al., (1996) Science 273: 1853-1856). Second, lsd1 plantsare resistant to normally virulent pathogens (Dietrich et al., (1997)Cell 88: 685-694). Thus, LSD1 is formally a negative regulator of RCDand of basal disease resistance. The deduced LSD1 protein is small (189amino acids), contains three highly related zinc-fingers, and canfunction as either a transcriptional regulator or as a scaffold protein(Dietrich et al., (1997) Cell 88: 685-694).

However, characterization of programmed cell death control in plantsremains a long-felt and continuing need in the art. The presentinvention addresses the need for further characterization of programmedcell death in plants, as well as other needs in the art.

SUMMARY OF THE INVENTION

Disclosed herein is an isolated and purified biologically active LOL1polypeptide that acts as a positive regulator of programmed cell deathin a plant. In one embodiment, the polypeptide comprises an amino acidsequence selected from the group consisting of SEQ ID NOs: 2 and 4-12,sequences having at least 85% identity with one of SEQ ID NOs: 2 and4-12, and fragments thereof. A chimeric polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs: 2 and4-12 is also disclosed. Optionally, a polypeptide of the presentinvention is in a detectably labeled form. An antibody that selectivelyrecognizes a polypeptide of the present invention is also provided.

Also disclosed herein is an isolated and purified nucleic acid sequenceencoding a LOL1 polypeptide that acts as a positive regulator ofprogrammed cell death in a plant. In one embodiment, the nucleic acidsequence comprises a nucleic acid sequence selected from the groupconsisting of: (a) SEQ ID NO: 1; (b) a sequence encoding a polypeptidecomprising a an amino acid sequence selected from the group consistingof SEQ ID NOs: 2 and 4-12; and (c) a nucleic acid sequence capable ofhybridizing under stringent conditions to a nucleic acid sequenceaccording to (a) or (b). The nucleic acid sequence can be a DNAsequence.

A chimeric gene comprising a nucleic acid sequence as disclosed hereinoperatively linked to a promoter is also provided, as is a recombinantvector comprising the chimeric gene. A host cell stably transformed withthe recombinant vector is also provided, as is a plant stablytransformed with the recombinant vector, along with a seed, progenyand/or part thereof. Optionally, the nucleic acid sequence is present inthe genome in a copy number effective to confer expression in the plantof a LOL1 polypeptide that acts as a positive regulator of programmedcell death in a plant. Also provided is a method of increasing LOL1 geneexpression in a plant, where the method comprises transforming the plantwith the recombinant vector. Also provided is a method of enhancingdisease resistance in a plant, where the method comprises transforming aplant with the recombinant vector.

Optionally, the nucleic acid sequence is expressed in the plant athigher levels than in a wild type plant. Also optionally, the plant isselected from the group consisting of Arabidopsis, rice, wheat, barley,rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory,lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach,asparagus, onion, garlic, eggplant, pepper, celery, squash, pumpkin,cucumber, apple, pear, quince, melon, plum, cherry, peach, nectarine,apricot, strawberry, grape, raspberry, blackberry, pineapple, avocado,papaya, mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.

A method of detecting a nucleic acid molecule that encodes a LOL1polypeptide that acts as a positive regulator of programmed cell deathin a plant in a biological sample containing nucleic acid material isalso provided. In one embodiment, the method comprises: (a) hybridizinga nucleic acid sequence as disclosed herein to nucleic acid material ofa biological sample under stringent hybridization conditions, to form ahybridization duplex; and (b) detecting the hybridization duplex.Optionally, the detected nucleic acid molecule further comprises achromosome.

A method of identifying positive regulation of programmed cell death ina plant is also provided. In one embodiment, the method comprises: (a)contacting a query nucleic acid sequence derived from a plant with aprobe comprising a nucleic acid sequence as disclosed herein; and (b)detecting the formation of a hybridized structure comprising the probeand the query nucleic acid sequence, the presence of a hybridizedstructure being indicative of positive regulation of programmed celldeath in the plant.

An assay kit for detecting the presence of a LOL1 polypeptide that actsas a positive regulator of programmed cell death in a plant is alsodisclosed. In one embodiment, the assay kit comprises a first containercontaining a nucleic acid probe comprising a sequence of ten or morecontiguous nucleotide bases corresponding to a fragment of a nucleicacid sequence as disclosed herein. Optionally, the kit further comprisesa second container containing a detectable moiety.

Accordingly, it is an object of the present invention to provide a novelpositive regulator of programmed cell death in plants. This and otherobjects are achieved in whole or in part by the present invention.

An object of the invention having been stated hereinabove, other objectswill be evident as the description proceeds, when taken in connectionwith the accompanying Drawings and Examples as best describedhereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the genomic sequence and deduced amino acid sequence ofthe A. thaliana LOL1 gene. The amino acid sequence is shown inone-letter-code. The stop codon is indicated by an asterisk (*). Aputative TATA-box is underlined. The cysteine residues of the three zincfinger domains are printed in bold and underlined. The position of theT-DNA insertion in the lol1-1 mutant (AFGC) is indicated by a triangle.

FIGS. 2A-2B shows the results of a comparison of the amino acid sequenceof the LOL1 protein from various monocotyledonous and dicotyledonousplants.

FIG. 2A shows a comparison between the conserved zinc finger motifs ofLSD1 and LOL1. Residues conserved in between three LSD1 and three LOL1zinc fingers are marked in bold. X constitutes any amino acid.

FIG. 2B depicts the deduced LOL1 amino acid sequence of ESTs from Zeamays (SEQ ID NO: 5), Sorghum propinquum (SEQ ID NO: 6), Oryza sativa(SEQ ID NO: 7), Triticum aestivum (SEQ ID NO: 8), Arabidopsis thaliana(SEQ ID NO: 9), Medicago trunculata (SEQ ID NO: 10), Solanum tuberosum(SEQ ID NO: 11), and Lycopersicon esculentum (SEQ ID NO: 12). Identitiesare shaded in black. Percent protein identities to Arabidopsis LOL1 andthe GenBank accession numbers (in some cases, the sequence of two ESTswere combined to generate the full length protein sequence) are given atthe end of each line. M, monocotyledonous species; D, dicotyledonousspecies. Dashes have been added to simplify the visual depiction of thealignment.

FIGS. 3A and 3B depict the results of treating Arabidopsis plants ofvarious genetic backgrounds with BTH.

FIG. 3A shows representative pictures of leaves taken at 7 days afterinoculation with 300 μM BTH. The genotypes are indicated above theleaves and from left to right are are as follows: Ws-0; lsd1;lsd/lol1-1; lsd1/lol1-as (clone 9); lsd1/LOL1-s (clone 4); lsd1/LOL1-s(clone 5).

FIG. 3B depicts the conductivity of leaves from 48 to 102 hours afterBTH treatment. The symbols represent the genotypes indicated at to theright of the graph and are as follows: lsd1/LOL1-s clone 4 (opensquare); lsd1 (open circle); lsd1/LOL1-s clone 5 (open triangle);lsd/lol1-1 (solid triangle); lsd1/lol1-as clone 9 (solid square); Ws-0(solid circle).

FIGS. 4A and 4B depicts the results of inoculating Arabidopsis plants ofvarious genetic backgrounds with Botiytis cinerea isolate A-1-3.

FIG. 4A shows representative pictures of leaves stained with lactophenoltrypan blue 3 days after inoculation. The genotypes are indicated abovethe leaves and from left to right are as follows: Ws-0; lsd1;lsd/lol1-1; lsd1/lol1-as (clone 9); lsd1/LOL1-s (clone 4); lsd1/LOL1-s(clone 5).

FIG. 4B depicts the average lesion size as determined by measuring theextent of lactophenol trypan blue staining on each leaf with a caliper.Key: Ws-0, black bar; lsd1, hatched bar; lsd1/lol1-as, gray bars (clones9 and 10); lsd1/LOL1-s, white bars (clones 4 and 5).

FIG. 5 is a graph of the conductivity of leaves immediately afterinfiltration with a 10 mM MgCl₂ solution containing Pst DC3000 (avrRpm1)at a concentration of 5×10⁷ cfu/ml. Control leaves infiltrated with 10mM MgCl₂ did not show HR or increased conductivity. Genotypes areindicated to the right of the graph, and the symbols are as follows:LOL1-s clone 5 (solid square); LOL1-s clone 9 (solid triangle); Ws-0(solid circle); lol1-as clone 9 (open triangle); lol1-1 (open square).

FIGS. 6A and 6B depict the results of spraying plants with P. parasiticaisolate Emco5 (1×10⁴ spores/ml).

FIG. 6A shows images at 100× magnification of inoculated leaves stainedwith lactophenol trypan blue 5 days after inoculation. Pictures weretaken with a Nikon ECLIPSE™E800 microscope with attached digital camera.Arrows indicate hyphae (Ws-0; left panel) and HR-like sites (LOL1-sclone 10; right panel).

FIG. 6B depicts susceptibility of the various plants as quantified bydetermining the number of spores produced on each genotype. Genotypesare indicated at the bottom of the graph, with line numbers designatedfor each genotype. From left to right the genotypes are Ws-0, white bar;lsd/lol1-1, lsd1/lol1-as (clone 9), gray bars; lsd1/LOL1-s, black bars(clones 3, 5, 7 and 10).

BRIEF DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING

SEQ ID NO: 1 is the genomic sequence of the Arabidopsis LOL1 gene.Depicted within the 3000 nucleotide genomic sequence is the deduced 154amino acid sequence of the LOL1 protein, including the positions of thefive coding exons.

SEQ ID NO: 2 is the deduced amino acid sequence of the Arabidopsis LOL1protein.

SEQ ID NO: 3 depicts the zinc finger consensus sequence from theArabidopsis LSD1 gene. In this sequence, non-conserved amino acids aredenoted by “Xaa”, while leucine¹, arginine⁷, serine¹⁷, and valine³⁰ aresemi-conserved.

SEQ ID NO: 4 depcits the zing finger consensus sequence from theArabodopsis LOL1 gene. In this sequence, non-conserved amino acids aredenoted by “Xaa”, while leucine⁹, serine¹⁸, and valine¹⁷ aresemi-conserved.

SEQ ID NOs: 5-12 are the amino acid sequences encoded by the LOL1 genefrom Zea mays (SEQ ID NO: 5), Sorghum propinquum (SEQ ID NO: 6), Oryzasativa (SEQ ID NO: 7), Triticum aestivum (SEQ ID NO: 8), Arabidopsisthaliana (SEQ ID NO: 9), Medicago trunculata (SEQ ID NO: 10), Solanumtuberosum (SEQ ID NO: 11), and Lycopersicon esculentum (SEQ ID NO: 12).

SEQ ID NOs: 13-15 are the sequences of primers that can be employedtogether in a polymerase chain reaction to detect the Arabidopsis lsd1mutation disclosed in the Examples.

SEQ ID NOs: 16 and 17 are the sequences of primers that can be usedtogether in a polymerase chain reaction to detect T-DNA insertion lol1-1into the Arabidopsis LOL1 gene, as disclosed in the Examples.

SEQ ID NOs: 18 and 19 are primers that can be used in a reversetranscription-PCR reaction to amplify Arabidopsis LOL1 gene transcriptsin order determine the relative mRNA levels of LOL1 present in thevarious antisense transgenic lines of the present invention, asdisclosed in the Examples.

DETAILED DESCRIPTION OF THE INVENTION

Programmed cell death control in plants is poorly understood. Thepresent invention comprises a novel, positive regulator of cell death inplants, such as Arabidopsis. This gene, LOL1, is a homologue of apreviously defined negative regulator of plant cell death called LSD1.Both encode small zinc finger proteins. The deduced LOL1 protein isremarkably conserved over 170-235 million years of evolution. LOL1positively regulates cell death in both an lsd1 mutant poised to undergorunaway cell death and in wild type plants challenged with pathogens.Modest over-expression of LOL1 led to significantly enhanced diseaseresistance against a virulent pathogen. The present invention thusconcerns in part the observation that this gene family plays a role inplant cell death control.

The present invention also concerns transformation vectors and processesfor expressing the above-noted genes in plants. The transgenic plantsthus created have, among other properties, disease resistance andpositive regulation of programmed cell death.

A chimeric gene comprising a promoter active in plants operativelylinked to a nucleic acid molecule encoding one of the above-noted genesis also disclosed, as is a recombinant vector comprising the chimericgene. Host cells stably transformed with the recombinant vector are alsodisclosed, as is a plant stably transformed with the recombinant vector.

A method of modulating gene expression in a plant, comprisingtransforming the plant with the recombinant vector is disclosed. Amethod of modulating disease resistance in a plant, which, in oneembodiment, comprises transforming the plant with the recombinantvector, is also disclosed.

A more detailed description of these and other aspects of the presentinvention is presented herein. Variations on these aspects of theinvention, as well as modification to the described aspects of theinvention will be apparent to those of ordinary skill in the art uponconsideration of the present disclosure; such modifications andvariations are within the scope of the present invention.

I. Definitions

Following long-standing patent law convention, the terms “a” and “an”mean “one or more” when used in this application, including the claims.

As used herein, the term “mutation” carries its traditional connotationand means a change, inherited, naturally occurring or introduced, in anucleic acid or polypeptide sequence, and is used in its sense asgenerally known to those of skill in the art.

As used herein, the term “transcription” means a cellular processinvolving the interaction of an RNA polymerase with a gene that directsthe expression as RNA of the structural information present in thecoding sequences of the gene. The process includes, but is not limitedto the following steps: (a) the transcription initiation, (b) transcriptelongation, (c) transcript splicing, (d) transcript capping, (e)transcript termination, (f) transcript polyadenylation, (g) nuclearexport of the transcript, (h) transcript editing, and (i) stabilizingthe transcript.

As used herein, the term “expression” generally refers to the cellularprocesses by which a polypeptide is produced from RNA.

As used herein, the term “hybridization” means the binding of a probemolecule, a molecule to which a detectable moiety has been bound, to atarget sample.

As used herein, the term “sequencing” means determining the orderedlinear sequence of nucleic acids or amino acids of a DNA or proteintarget sample, using conventional manual or automated laboratorytechniques.

As used herein, the term “isolated” means oligonucleotides substantiallyfree of other nucleic acids, proteins, lipids, carbohydrates or othermaterials with which they can be associated, such association beingeither in cellular material or in a synthesis medium. The term can alsobe applied to polypeptides, in which case the polypeptide will besubstantially free of nucleic acids, carbohydrates, lipids and otherundesired polypeptides.

As used herein, the term “substantially pure” means that thepolynucleotide or polypeptide is substantially free of the sequences andmolecules with which it is associated in its natural state, and thosemolecules used in the isolation procedure. The term “substantially free”means that the sample is at least 50%, preferably at least 70%, morepreferably 80% and most preferably 90% to 99% free of the materials andcompounds with which is it associated in nature.

As used herein, the term “primer” means a sequence comprising, forexample, two or more deoxyribonucleotides or ribonucleotides, more thanthree deoxyribonucleotides or ribonucleotides, more than eightdeoxyribonucleotides or ribonucleotides or at least about 20deoxyribonucleotides or ribonucleotides of an exonic or intronic region.Such oligonucleotides can be, for example, between ten and thirty basesin length.

As used herein, the term “DNA segment” means a DNA molecule that hasbeen isolated free of total genomic DNA of a particular species. In oneembodiment, a DNA segment encoding a LOL1 polypeptide refers to a DNAsegment that comprises SEQ ID NO: 1, but can optionally comprise feweror additional nucleic acids, yet is isolated away from, or purified freefrom, total genomic DNA of a source species, such as Arabisopsis.Included within the term “DNA segment” are DNA segments and smallerfragments of such segments, and also recombinant vectors, including, forexample, plasmids, cosmids, phages, viruses, and the like.

As used herein, the phrase “enhancer-promoter” means a composite unitthat contains either or both enhancer and promoter elements. Anenhancer-promoter is operatively linked to a coding sequence thatencodes at least one gene product.

As used herein, the phrase “operatively linked” means that anenhancer-promoter is connected to a coding sequence in such a way thatthe transcription of that coding sequence is controlled and regulated bythat enhancer-promoter. Techniques for operatively linking anenhancer-promoter to a coding sequence are well known in the art; theprecise orientation and location relative to a coding sequence ofinterest can be dependent, inter alia, upon the specific nature of theenhancer-promoter.

As used herein, the term “biological activity” means any observableeffect flowing from a LOL1 polypeptide. Representative, butnon-limiting, examples of biological activity in the context of thepresent invention include disease resistance and modulation of PCD.

As used herein, the term “modified” means an alteration from an entity'snormally occurring state. An entity can be modified by removing discretechemical units or by adding discrete chemical units. The term “modified”encompasses detectable labels as well as those entities added as aids inpurification.

As used herein, the term “LOL1” means nucleic acids encoding afunctional LOL1 polypeptide. The term “LOL1” includes homologs.

As used herein, the terms “LOL1 gene product”, “LOL1 protein”, “LOL1polypeptide”, and “LOL1 peptide” are used interchangeably and meanpeptides having amino acid sequences which are substantially identicalto native amino acid sequences from an organism of interest and whichare biologically active in that they comprise all or a part of the aminoacid sequence of a LOL1 polypeptide, or cross-react with antibodiesraised against a LOL1 polypeptide, or retain all or some of thebiological activity (e.g., regulation of cell death and/or diseaseresistance) of the native amino acid sequence or protein. Suchbiological activity can include immunogenicity.

As used herein, the terms “LOL1 gene product”, “LOL1 protein”, “LOL1polypeptide”, and “LOL1 peptide” also include analogs of a LOL1polypeptide. By “analog” is intended that a DNA or peptide sequence cancontain alterations relative to the sequences disclosed herein, yetretain all or some of the biological activity of those sequences.Analogs can be derived from genomic nucleotide sequences as aredisclosed herein or from other organisms, or can be createdsynthetically. Those skilled in the art will appreciate that otheranalogs, as yet undisclosed or undiscovered, can be used to designand/or construct LOL1 analogs. There is no need for a “LOL1 geneproduct”, “LOL1 protein”, “LOL1 polypeptide”, or “LOL1 peptide” tocomprise all or substantially all of the amino acid sequence of a LOL1polypeptide gene product. Shorter or longer sequences are anticipated tobe of use in the invention; shorter sequences are herein referred to as“segments”. Thus, the terms “LOL1 gene product”, “LOL1 protein”, “LOL1polypeptide”, and “LOL1 peptide” also include fusion, chimeric orrecombinant LOL1 polypeptides and proteins comprising sequences of thepresent invention. Methods of preparing such proteins are disclosedherein and are known in the art.

As used herein, the term “polypeptide” means any polymer comprising anyof the 20 protein amino acids, regardless of its size. Although“protein” is often used in reference to relatively large polypeptides,and “peptide” is often used in reference to small polypeptides, usage ofthese terms in the art overlaps and varies. The term “polypeptide” asused herein refers to peptides, polypeptides and proteins, unlessotherwise noted. As used herein, the terms “protein”, “polypeptide” and“peptide” are used interchangeably herein when referring to a geneproduct.

As used herein, the terms “LOL1 gene” and “recombinant LOL1 gene” mean anucleic acid molecule comprising an open reading frame encoding a LOL1polypeptide of the present invention, including both exon and(optionally) intron sequences.

The term “gene” refers broadly to any segment of DNA associated with abiological function. A gene encompasses sequences including but notlimited to a coding sequence, a promoter region, a cis-regulatorysequence, a non-expressed DNA segment is a specific recognition sequencefor regulatory proteins, a non-expressed DNA segment that contributes togene expression, a DNA segment designed to have desired parameters, orcombinations thereof. A gene can be obtained by a variety of methods,including cloning from a biological sample, synthesis based on known orpredicted sequence information, and recombinant derivation of anexisting sequence.

As used herein, the term “DNA sequence encoding a LOL1 polypeptide” canrefer to one or more coding sequences within a particular individual.Moreover, certain differences in nucleotide sequences can exist betweenindividual organisms, which are called alleles. It is possible that suchallelic differences might or might not result in differences in aminoacid sequence of the encoded polypeptide yet still encode a protein withthe same biological activity. As is well known, genes for a particularpolypeptide can exist in single or multiple copies within the genome ofan individual. Such duplicate genes can be identical or can have certainmodifications, including nucleotide substitutions, additions ordeletions, all of which still code for polypeptides having substantiallythe same activity.

As used herein, the term “intron” means a DNA sequence present in agiven gene that is not translated into protein.

As used herein, the terms “cells,” “host cells” or “recombinant hostcells” are used interchangeably and mean not only to the particularsubject cell, but also to the progeny or potential progeny of such acell. Because certain modifications can occur in succeeding generationsdue to either mutation or environmental influences, such progeny mightnot, in fact, be identical to the parent cell, but are still includedwithin the scope of the term as used herein.

As used herein, the terms “chimeric protein” or “fusion protein’ areused interchangeably and mean a fusion of a first amino acid sequenceencoding a LOL1 polypeptide with a second amino acid sequence defining apolypeptide domain foreign to, and not homologous with, a LOL1polypeptide. A chimeric protein can present a foreign domain that isfound in an organism that also expresses the first protein, or it can bean “interspecies” or “intergenic” fusion of protein structures expressedby different kinds of organisms. In general, a fusion protein can berepresented by the general formula X-LOL1-Y, wherein LOL1 represents aportion of the protein which is derived from a LOL1 polypeptide, and Xand Y are independently absent or represent amino acid sequences whichare not related to a LOL1 sequence in an organism, which includesnaturally occurring mutants. Analogously, the term “chimeric gene”refers to a nucleic acid construct that encodes a “chimeric protein” or“fusion protein” as defined herein.

As used herein, the term “recombination” and grammatical derivationsthereof, means a re-assortment of genes or characters in combinationsdifferent from what they were in the parents, in the case of linkedgenes by crossing over.

As used herein, the term “plant” means an entire plant. The term “partof a plant” means the individual parts of a plant, including but notlimited to seeds, leaves, stems and roots, as well as plant tissuecultures.

As used herein the term “complementary” means a nucleic acid sequencethat is capable of base-pairing according to the standard Watson-Crickcomplementarity rules. That is, that the larger purines will always basepair with the smaller pyrimidines to form only combinations of Guaninepaired with Cytosine (G:C) and Adenine paired with either Thymine (A:T)in the case of DNA or Adenine paired with Uracil (A:U) in the case ofRNA.

As used herein, the term “hybridization techniques” refers to molecularbiological techniques that involve the binding or hybridization of aprobe to complementary sequences in a polynucleotide. Included amongthese techniques are northern blot analysis, Southern blot analysis,nuclease protection assay, etc.

As used herein, the terms “hybridization” and “binding” are usedinterchangeably in the context of probes and denatured DNA. Probes thatare hybridized or bound to denatured DNA are aggregated to complementarysequences in the polynucleotide. Whether or not a particular proberemains aggregated with the polynucleotide depends on the degree ofcomplementarity, the length of the probe, and the stringency of thebinding conditions. The higher the stringency, the higher must be thedegree of complementarity and/or the longer the probe.

As used herein, the term “probe” refers to an oligonucleotide or shortfragment of DNA designed, known or suspected to be sufficientlycomplementary to a sequence in a denatured nucleic acid to be probed andto be bound under selected stringency conditions.

As used herein, the term “cloning” means separation and/or isolation ofgenes.

As used herein, the term “stringent hybridization conditions”, in thecontext of nucleic acid hybridization experiments such as Southern andNorthern blot analysis, means a set of conditions under which singlestranded nucleic acid sequences are unlikely to hybridize to one anotherunless there is substantial complementarity between the sequences.Stringent hybridization conditions can be both sequence- andenvironment-dependent. Longer sequences hybridize specifically at highertemperatures. An extensive guide to the hybridization of nucleic acidsis found in Tijssen, (1993) Laboratory Techniques in Biochemistry andMolecular Biology-Hybridization with Nucleic Acid Probes, part 1,chapter 2, Elsevier, New York. Generally, highly stringent hybridizationand wash conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence at a definedionic strength and pH. Typically, under “stringent conditions” a probewill hybridize specifically to its target subsequence, but to no othersequences.

The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe. Very stringent conditions are selected to be equal to the T_(m)for a particular probe. An example of stringent hybridization conditionsfor Southern or Northern Blot analysis of complementary nucleic acidshaving more than about 100 complementary residues is overnighthybridization in 50% formamide with 1 mg of heparin at 42° C. An exampleof highly stringent wash conditions is 15 minutes in 0.15 M NaCl at 65°C. An example of stringent wash conditions is 15 minutes in 0.2×SSCbuffer at 65° C. (See Sambrook (1989) for a description of SSC buffer).Often, a high stringency wash is preceded by a low stringency wash toremove background probe signal. An example of medium stringency washconditions for a duplex of more than about 100 nucleotides, is 15minutes in 1×SSC at 45° C. An example of low stringency wash for aduplex of more than about 100 nucleotides, is 15 minutes in 4-6×SSC at40° C. For short probes (e.g., about 10 to 50 nucleotides), stringentconditions typically involve salt concentrations of less than about 1.0M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or othersalts) at pH 7.0-8.3, and the temperature is typically at least about30° C. Stringent conditions can also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2-fold (or higher) than that observed for an unrelated probe ina particular hybridization assay indicates the presence of a specifichybridization.

The following are examples of hybridization and wash conditions that canbe used to clone homologous nucleotide sequences that are substantiallyidentical to reference nucleotide sequences of the present invention: aprobe nucleotide sequence preferably hybridizes to a target nucleotidesequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. followed by washing in 2×SSC, 0.1% SDS at 50° C.; morepreferably, a probe and target sequence hybridize in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. followed by washing in 1×SSC, 0.1% SDS at 50° C.; more preferably, a probe and target sequencehybridize in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at50° C. followed by washing in 0.5×SSC, 0.1% SDS at 50° C.; morepreferably, a probe and target sequence hybridize in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. followed by washing in0.1×SSC, 0.1% SDS at 50° C.; more preferably, a probe and targetsequence hybridize in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. followed by washing in 0.1×SSC, 0.1% SDS at 65° C.

As used herein, the term “gene expression” means the cellular processesby which a biologically active polypeptide is produced from a DNAsequence.

As used herein, the term “vector” means a DNA molecule having sequencesthat enable its replication in a compatible host cell. A vector alsoincludes nucleotide sequences to permit ligation of nucleotide sequenceswithin the vector, wherein such nucleotide sequences are also replicatedin a compatible host cell. A vector can also mediate recombinantproduction of an LOL1 polypeptide, as described further herein below.Some representative vectors include, but are not limited to, pBluescript(Stratagene), pUC18, pBLCAT3 (Luckow and Schutz, (1987) Nucleic AcidsRes 15: 5490), pLNTK (Gorman et al., (1996) Immunity 5: 241-252), andpBAD/gIII (Stratagene). A representative host cell is an Arabidopsiscell.

II. General Considerations

Zinc finger modules are approximately 30 amino acid-long motifs found ina wide variety of transcription regulatory proteins in eukaryoticorganisms. As the name implies, this nucleic acid binding protein domainis folded around a zinc ion. The zinc finger domain was first recognizedin the transcription factor TFIIIA from Xenopus oocytes (Miller et al.,(1985) EMBO 4:1609-1614; Brown et al., (1985) FEBS Lett. 186:271-274).

Families of zinc finger proteins often function in related processes.For example, the mammalian GATA family of transcription factors isimportant in erythroid and embryonic development (Charron et al., (1999)Mol. Cell Biol. 19: 4355-4365; Molkentin, (2000) J. Bio. Chem. 275:38949-38952; Pevny et al., (1991) Nature 349: 257-260), and themammalian IAP protein family negatively controls PCD (Deveraux & Reed,(1999) Genes Dev. 13: 239-252; Miller, (1999) Trends Cell Bio. 9:323-328; Verhagen et al., (2001) Genome Biol. 2).

Transcriptional regulation is primarily achieved by thesequence-specific binding of proteins to DNA and RNA. Of the knownprotein motifs involved in the sequence specific recognition of DNA, thezinc finger protein is unique in its modular nature. To date, zincfinger proteins have been identified which contain between 2 and 37modules. More than two hundred proteins, many of them transcriptionfactors, have been shown to possess zinc fingers domains. Zinc fingersconnect transcription factors to their target genes mainly by binding tospecific sequences of DNA base pairs—the “rungs” in the DNA “ladder”.

Systemic acquired resistance (SAR) is one component of the complexsystem plants use to defend themselves from pathogens (Hunt & Ryals,(1996) Crit. Rev. in Plant Sci. 15, 583-606; Ryals et al., (1996) PlantCell 8, 1809-1819). See also U.S. Pat. No. 5,614,395. SAR is an aspectof plant-pathogen responses because it is a pathogen-inducible, systemicresistance against a broad spectrum of infectious agents, includingviruses, bacteria, and fungi. When the SAR signal transduction pathwayis blocked, plants become more susceptible to pathogens that normallycause disease, and they also become susceptible to some infectiousagents that would not normally cause disease (Gaffney et al., (1993)Science 261: 754-756; Delaney et al., (1994) Science 266: 1247-1250;Delaney et al., (1995) Proc. Natl. Acad. Sci. USA 92: 6602-6606;Delaney, (1997) Plant Phys. 113: 5-12; Bi et al., (1995) Plant J. 8:235-245; Mauch-Mani & Slusarenko, (1996) Plant Cell 8: 203-212). Theseobservations indicate that the SAR signal transduction pathway plays arole in maintaining plant health.

Salicylic acid (SA) accumulation appears to be required for SAR signaltransduction. Plants that cannot accumulate SA due to treatment withspecific inhibitors, epigenetic repression of phenylalanineammonia-lyase, or transgenic expression of salicylate hydroxylase, whichspecifically degrades SA, also cannot induce either SAR gene expressionor disease resistance (Gaffney et al., (1993) Science 261, 754-756;Delaney et al., (1994) Science 266, 1247-1250; Mauch-Mani & Slusarenko,(1996) Plant Cell 8: 203-212; Maher et al., (1994) Proc. Natl. Acad.Sci. USA 91: 7802-7806; Pallas et al., (1996) Plant J. 10: 281-293).Although it has been suggested that SA might serve as the systemicsignal, this is currently controversial and, to date, all that is knownfor certain is that if SA cannot accumulate, then SAR signaltransduction is blocked (Pallas et al., (1996) Plant J. 10: 281-293;Shulaey et al., (195) Plant Cell 7, 1691-1701; Vernooij et al., (1994)Plant Cell 6: 959-965).

Arabidopsis has emerged as a model system to study SAR (Uknes et al.,(1992) Plant Cell 4: 645-656, incorporated by reference herein in itsentirety; Uknes et al., (1993) Mol. Plant-Microbe Interact 6: 692-698;Cameron et al., (1994) Plant J. 5: 715-725; Mauch-Mani & Slusarenko,(1994) Mol. Plant-Microbe Interact 7: 378-383; Dempsey & Klessig, (1995)Bulletin de L'Institut Pasteur 93: 167-186). It has been demonstratedthat SAR can be activated in Arabidopsis by both pathogens andchemicals, such as SA, 2,6-dichloroisonicotinic acid (INA) andbenzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) (Ukneset al., (1992) Plant Cell 4: 645-656; Vernooij et al., (1995) Mol.Plant-Microbe Interact. 8: 228-234; Lawton et al., (1996) Plant J. 10:71-82). Following treatment with either INA or BTH or pathogeninfection, at least three pathogenesis-related (PR) protein genes,namely, PR-1, PR-2, and PR-5 are coordinately induced concomitant withthe onset of resistance. In tobacco, the best characterized species,treatment with a pathogen or an immunization compound induces theexpression of at least nine sets of genes (Ward et al., (1991) PlantCell 3: 1085-1094). Transgenic disease-resistant plants have beencreated by transforming plants with various SAR genes (U.S. Pat. No.5,614,395).

A number of Arabidopsis mutants have been isolated that have modifiedSAR signal transduction (Delaney, (1997) Plant Phys. 113: 5-12). Thefirst of these mutants are the so-called lsd (lesions simulatingdisease) mutants and acd2 (accelerated cell death) (Dietrich et al.(1994) Cell 77: 565-577; Greenberg et al., (1994) Cell 77: 551-563).These mutants all have some degree of spontaneous necrotic lesionformation on their leaves, elevated levels of SA, mRNA accumulation forthe SAR genes, and significantly enhanced disease resistance. At leastseven different lsd mutants have been isolated and characterized(Dietrich et al., (1994) Cell 77: 565-577; Weymann et al., (1995) PlantCell 7: 2013-2022).

III. Production of LOL1 Polypeptides

The native and mutated LOL1 polypeptides, and fragments thereof, of thepresent invention can be chemically synthesized in whole or part usingtechniques that are well-known in the art (see, e.g., Creighton, (1983)Proteins: Structures and Molecular Principles, W. H. Freeman & Co., NewYork). Alternatively, methods that are well known to those skilled inthe art can be used to construct expression vectors containing a partialor the entire native or mutated LOL1 polypeptide coding sequence andappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, as described herein,synthetic techniques and in vivo recombination/genetic recombination(see, e.g., the techniques described in Sambrook et al., (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, and Ausubel et al., (1992) Current Protocols in MolecularBiology John Wylie and Sons, Inc. New York, both incorporated herein intheir entirety).

IV. Recombinant DNA Technology

Nucleic acids of the present invention can be cloned, synthesized,recombinantly altered, mutagenized, or combinations thereof. Standardrecombinant DNA and molecular cloning techniques used to isolate nucleicacids are well known in the art. Exemplary, non-limiting methods aredescribed by, for example, Sambrook et al., (eds.) (1989) MolecularCloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NewYork; by Silhavy et al., (1984) Experiments with Gene Fusions, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, New York; by Ausubelet al., (1992) Current Protocols in Molecular Biology John Wylie andSons, Inc. New York; and by Glover, (ed.) (1985) DNA Cloning: APractical Approach, MRL Press, Ltd., Oxford, U.K. Site-specificmutagenesis to create base pair changes, deletions, or small insertionsare also well known in the art as exemplified by publications (see,e.g., Adelman et al., (1983) DNA 2:183; Sambrook et al., (eds.) (1989)Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, New York).

Sequences disclosed or detected by methods of the invention candetected, subcloned, sequenced, and further evaluated by any measurewell known in the art using any method usually applied to the detectionof a specific DNA sequence including but not limited to dideoxysequencing, PCR, oligomer restriction (Saiki et al., (1985)Bio/Technology 3: 1008-1012, allele-specific oligonucleotide (ASO) probeanalysis (Conner et al., (1983) Proc. Natl. Acad. Sci. U.S.A. 80: 278),and oligonucleotide ligation assays (OLAs) (Landgren et. al., (1988)Science 241: 1007). Molecular techniques for DNA analysis have beenreviewed (Landgren et. al., (1988) Science 242: 229-237).

Thus, the LOL1 genes disclosed herein can be incorporated in plant orbacterial cells using conventional recombinant DNA technology.Generally, this involves inserting DNA molecule encoding an LOL1polypeptide as described herein into an expression system to which theDNA molecule is homologous (i.e., normally present) however a system canalso be heterologous (i.e., not normally present). This insertion can bemade using standard cloning procedures known in the art. A sutiablevector can contain the necessary elements for the transcription andtranslation of the inserted protein-coding sequences. A large number ofvector systems known in the art can be used, such as plasmids,bacteriophage viruses and other modified viruses. Suitable vectorsinclude, but are not limited to, viral vectors such as lambda vectorsystems λgt11, λgt10 and Charon4; plasmid vectors such as pBI121,pBR322, pACYC177, pACYC184, pAR series, pKK223-3, pUC8, pUC9, pUC18,pUC19, pLG339, pRK290, pKC37, pKC101, pCDNAII; and other similarsystems.

The components of an expression system can also be modified to increaseexpression. For example, truncated sequences, nucleotide substitutionsor other modifications can be employed. The expression systems describedherein can be used to transform virtually any crop plant cell undersuitable conditions. Transformed cells can be regenerated into wholeplants such that the chosen form of the LOL1 gene is expressed in thetransgenic plants.

IV.A. Construction of Plant Expression Cassettes

In one example, gene sequences intended for expression in transgenicplants can be assembled in expression cassettes behind a suitablepromoter expressible in plants. An expression cassette can also compriseany further sequences required or selected for the expression of thetransgene. Such sequences include, but are not restricted to,transcription terminators, extraneous sequences to enhance expressionsuch as introns, vital sequences, and sequences intended for thetargeting of the gene product to specific organelles and cellcompartments. These expression cassettes can then be transferred to theplant transformation vectors described herein. The following is adescription of some components of a typical expression cassette that canbe employed in the present invention.

IV.B. Promoters

The selection of the promoter used in an expression cassette candetermine the spatial and temporal expression pattern of the transgenein the transgenic plant. Selected promoters will express transgenes inspecific cell types (such as leaf epidermal cells, mesophyll cells, rootcortex cells) or in specific tissues or organs (roots, leaves orflowers, for example) and the selection will reflect the desiredlocation of accumulation of the gene product. Alternatively, theselected promoter can drive expression of the gene under variousinducing conditions. Promoters vary in their strength, i.e., ability topromote transcription. Depending upon the host cell system utilized, anyone of a number of suitable promoters can be used. The following arenon-limiting examples of promoters that can be used in the expressioncassettes.

IV.B.1. Constitutive Expression, the CaMV 35S Promoter

Construction of the plasmid pCGN1761 is described in the publishedpatent application EP 0 392 225, which is hereby incorporated byreference. pCGN1761 contains the “double” CaMV 35S promoter and the tmltranscriptional terminator with a unique EcoRI site between the promoterand the terminator and has a pUC-type backbone. A derivative of pCGN1761is constructed which has a modified polylinker that includes NotI andXhoI sites in addition to the existing EcoRI site. This derivative isdesignated pCGN1761ENX. pCGN1761ENX is useful for the cloning of cDNAsequences or gene sequences (including microbial ORF sequences) withinits polylinker for the purpose of their expression under the control ofthe 35S promoter in transgenic plants. The entire 35S promoter-genesequence-tml terminator cassette of such a construction can be excisedby HindIII, SphI, SaII, and XbaI sites 5′ to the promoter and XbaI,BamHI and BgII sites 3′ to the terminator for transfer to transformationvectors such as those described below. Furthermore, the double 35Spromoter fragment can be removed by 5′ excision with HindIII, SphI,SaII, XbaI, or PstI, and 3′ excision with any of the polylinkerrestriction sites (EcoRI, NotI or XhoI) for replacement with anotherpromoter.

If desired, modifications around the cloning sites can be made by theintroduction of sequences that can enhance translation. This isparticularly useful when overexpression of a LOL1 polypeptide isdesired. For example, pCGN1761ENX can be modified by optimization of thetranslational initiation site as disclosed in U.S. Pat. No. 5,639,949,incorporated herein by reference.

IV.B.2.Expression Under a Chemically/Pathogen Regulatable Promoter

The double 35S promoter in pCGN1761ENX can be replaced with any otherpromoter of choice, which will result in suitably high expressionlevels. By way of example, one of the chemically regulatable promotersdescribed in U.S. Pat. No. 5,614,395 can replace the double 35Spromoter. The promoter of choice is preferably excised from its sourceby restriction enzymes, but can alternatively be PCR-amplified usingprimers that carry appropriate terminal restriction sites. ShouldPCR-amplification be undertaken, the promoter should be re-sequenced tocheck for amplification errors after the cloning of the amplifiedpromoter in the target vector. The chemically/pathogen regulatabletobacco PR-1a promoter is cleaved from plasmid pCIB1004 (forconstruction, see EP 0 332 104, which is hereby incorporated byreference) and transferred to plasmid pCGN1761ENX (Uknes et al., (1992)Plant Cell 4: 645-656).

pCIB1004 is cleaved with NcoI and the resultant 3′ overhang of thelinearized fragment is rendered blunt by treatment with T4 DNApolymerase. The fragment is then cleaved with HindIII and the resultantPR-1a promoter-containing fragment is gel purified and cloned intopCGN1761ENX from which the double 35S promoter has been removed. This isdone by cleavage with XhoI and blunting with T4 polymerase, followed bycleavage with HindIII and isolation of the larger vector-terminatorcontaining fragment into which the pCIB1004 promoter fragment is cloned.This generates a pCGN1761ENX derivative with the PR-1a promoter and thetml terminator and an intervening polylinker with unique EcoRI and NotIsites. The selected coding sequence can be inserted into this vector,and the fusion products (i.e. promoter-gene-terminator) can subsequentlybe transferred to any selected transformation vector, including thosedescribed below. Various chemical regulators can be employed to induceexpression of the selected coding sequence in the plants transformedaccording to the present invention, including the benzothiadiazole,isonicotinic acid, and salicylic acid compounds disclosed in U.S. Pat.Nos. 5,523,311 and 5,614,395, herein incorporated by reference.

IV.B.3. Constitutive Expression, the Actin Promoter

Several isoforms of actin are known to be expressed in most cell typesand consequently the actin promoter is a good choice for a constitutivepromoter. In particular, the promoter from the rice ActI gene has beencloned and characterized (McElroy et al., (1990) Plant Cell 2: 163-171).A 1.3 kb fragment of the promoter was found to contain all theregulatory elements required for expression in rice protoplasts.Furthermore, numerous expression vectors based on the ActI promoter havebeen constructed specifically for use in monocotyledons (McElroy et al.,(1991) Mol. Gen. Genet. 231: 150-160). These incorporate the ActI-intron1, Adhl 5′ flanking sequence and Adhl-intron 1 (from the maize alcoholdehydrogenase gene) and sequence from the CaMV 35S promoter. Vectorsshowing highest expression were fusions of 35S and ActI intron or theActI 5′ flanking sequence and the ActI intron. Optimization of sequencesaround the initiating ATG (of the GUS reporter gene) also enhancedexpression. The promoter expression cassettes described by McElroy etal. (McElroy et al., (1991) Mol. Gen. Genet. 231: 150-160) can be easilymodified for gene expression and are particularly suitable for use inmonocotyledonous hosts. For example, promoter-containing fragments isremoved from the McElroy constructions and used to replace the double35S promoter in pCGN1761ENX, which is then available for the insertionof specific gene sequences. The fusion genes thus constructed can thenbe transferred to appropriate transformation vectors. In a separatereport, the rice ActI promoter with its first intron has also been foundto direct high expression in cultured barley cells (Chibbar et al.,(1993) Plant Cell Rep. 12: 506-509).

IV.B.4. Constitutive Expression, the Ubiguitin Promoter

Ubiguitin is another gene product known to accumulate in many cell typesand its promoter has been cloned from several species for use intransgenic plants (e.g. sunflower—Binet et al., (1991) Plant Science 79:87-94 and maize—Christensen et al., (1989) Plant Molec. Biol. 12:619-632). The maize ubiguitin promoter has been developed in transgenicmonocot systems and its sequence and vectors constructed for monocottransformation are disclosed in the patent publication EP 0 342 926which is herein incorporated by reference. Taylor et al. (Taylor et al.,(1993) Plant Cell Rep. 12: 491-495) describe a vector (pAHC25) thatcomprises the maize ubiguitin promoter and first intron and its highactivity in cell suspensions of numerous monocotyledons when introducedvia microprojectile bombardment. The ubiguitin promoter is suitable forgene expression in transgenic plants, especially monocotyledons.Suitable vectors are derivatives of pAHC25 or any of the transformationvectors described in this application, modified by the introduction ofthe appropriate ubiguitin promoter and/or intron sequences.

IV.B.5. Root Specific Expression

Another pattern of gene expression is root expression. A suitable rootpromoter is described by de Framond (de Framond, (1991) FEBS 290:103-106) and also in the published patent application EP 0 452 269,which is herein incorporated by reference. This promoter is transferredto a suitable vector such as pCGN1761ENX for the insertion of a selectedgene and subsequent transfer of the entire promoter-gene-terminatorcassette to a transformation vector of interest.

IV.B.6. Wound-Inducible Promoters

Wound-inducible promoters can also be suitable for gene expression.Numerous such promoters have been described (e.g. Xu et al., (1993)Plant Molec. Biol. 22: 573-588, Logemann et al., (1989) Plant Cell 1:151-158, Rohrmeier & Lehle, (1993) Plant Molec. Biol. 22: 783-792, Fireket al., (1993) Plant Molec. Biol. 22: 129-142, Warner et al., (1993)Plant J. 3: 191-201) and all are suitable for use with the instantinvention. Logemann et al. describe the 5′ upstream sequences of thedicotyledonous potato wunl gene. Xu et al. show that a wound-induciblepromoter from the dicotyledon potato (pin2) is active in themonocotyledon rice. Further, Rohrmeier & Lehle describe the cloning ofthe maize Wipl cDNA, which is wound induced and which can be used toisolate the cognate promoter using standard techniques. Similarly, Fireket al. and Warner et al. have described a wound-induced gene from themonocotyledon Asparagus officinalis, which is expressed at local woundand pathogen invasion sites. Using cloning techniques well known in theart, these promoters can be transferred to suitable vectors, fused tothe genes pertaining to this invention, and used to express these genesat the sites of plant wounding.

IV.B.7. Pith-Preferred Expression

Patent Application WO 93/07278, which is herein incorporated byreference, describes the isolation of the maize trpA gene, which ispreferentially expressed in pith cells. The gene sequence and promoterextending up to −1726 bp from the start of transcription are presented.Using standard molecular biological techniques, this promoter, or partsthereof, can be transferred to a vector such as pCGN1761 where it canreplace the 35S promoter and be used to drive the expression of aforeign gene in a pith-preferred manner. In fact, fragments containingthe pith-preferred promoter or parts thereof can be transferred to anyvector and modified for utility in transgenic plants.

IV.B.8. Leaf-Specific Expression

A maize gene encoding phosphoenol carboxylase (PEPC) has been describedby Hudspeth & Grula (Hudspeth & Grula, (1989) Plant Molec Biol 12:579-589). Using standard molecular biological techniques the promoterfor this gene can be used to drive the expression of any gene in aleaf-specific manner in transgenic plants.

IV.C. Transcriptional Terminators

A variety of transcriptional terminators are available for use inexpression cassettes. These are responsible for the termination oftranscription beyond the transgene and its correct polyadenylation.Appropriate transcriptional terminators are those that are known tofunction in plants and include the CaMV 35S terminator, the tmlterminator, the nopaline synthase terminator and the pea rbcS E9terminator. These can be used in both monocotyledons and dicotyledons.

IV.D. Sequences for the Enhancement or Regulation of Expression

Numerous sequences have been found to enhance gene expression fromwithin the transcriptional unit and these sequences can be used inconjunction with the genes of this invention to increase theirexpression in transgenic plants.

Various intron sequences have been shown to enhance expression,particularly in monocotyledonous cells. For example, the introns of themaize AdhI gene have been found to significantly enhance the expressionof the wild-type gene under its cognate promoter when introduced intomaize cells. Intron 1 was found to be particularly effective andenhanced expression in fusion constructs with the chloramphenicolacetyltransferase gene (Callis et al., (1987) Genes Develop. 1:1183-1200). In the same experimental system, the intron from the maizebronze1 gene had a similar effect in enhancing expression. Intronsequences have been routinely incorporated into plant transformationvectors, typically within the non-translated leader.

A number of non-translated leader sequences derived from viruses arealso known to enhance expression, and these are particularly effectivein dicotyledonous cells. Specifically, leader sequences from TobaccoMosaic Virus (TMV, the “W-sequence”), Maize Chlorotic Mottle Virus(MCMV), and Alfalfa Mosaic Virus (AMV) have been shown to be effectivein enhancing expression (e.g., Gallie et al., (1987) Nucl. Acids Res.15: 8693-8711; Skuzeski et al., (1990) Plant Molec. Biol. 15: 65-79).

IV.E. Targeting the Gene Product Within the Cell

Various mechanisms for targeting gene products are known to exist inplants and the sequences controlling the functioning of these mechanismshave been characterized in some detail. For example, the targeting ofgene products to the chloroplast is controlled by a signal sequencefound at the amino terminal end of various proteins, which is cleavedduring chloroplast import to yield the mature protein (see e.g., Comaiet al., (1988) J. Biol. Chem. 263: 15104-15109). These signal sequencescan be fused to heterologous gene products to effect the import ofheterologous products into the chloroplast (van den Broeck et al.,(1985) Nature 313: 358-363). DNA encoding for appropriate signalsequences can be isolated from the 5′ end of the cDNAs encoding theRUBISCO protein, the CAB protein, the EPSP synthase enzyme, the GS2protein and many other proteins which are known to be chloroplastlocalized. See also, U.S. Pat. No. 5,639,949, herein incorporated byreference.

Other gene products are localized to other organelles such as themitochondrion and the peroxisome (see, e.g., Unger et al., (1989) PlantMolec. Biol. 13: 411-418). The cDNAs encoding these products can also bemanipulated to effect the targeting of heterologous gene products tothese organelles. Examples of such sequences are the nuclear-encodedATPases and specific aspartate amino transferase isoforms formitochondria. Targeting cellular protein bodies has been described byRogers et al. (Rogers et al., (1985) Proc. Natl. Acad. Sci. USA 82:6512-6516).

In addition, sequences have been characterized which cause the targetingof gene products to other cell compartments. Amino terminal sequencesare responsible for targeting to the ER, the apoplast, and extracellularsecretion from aleurone cells (Koehler & Ho, (1990) Plant Cell 2:769-783). Additionally, amino terminal sequences in conjunction withcarboxy terminal sequences are responsible for vacuolar targeting ofgene products (Shinshi et al., (1990) Plant Molec. Biol. 14: 357-368).

By fusing an appropriate targeting sequences described above totransgene sequences of interest it is possible to direct the transgeneproduct to a given organelle or cell compartment. For chloroplasttargeting, for example, the chloroplast signal sequence from the RUBISCOgene, the CAB gene, the EPSP synthase gene, or the GS2 gene is fused inframe to the amino terminal ATG of the transgene. The signal sequenceselected should include the known cleavage site, and the fusionconstructed should take into account any amino acids after the cleavagesite that are required for cleavage. In some cases this requirement canbe fulfilled by the addition of a small number of amino acids betweenthe cleavage site and the transgene ATG or, alternatively, replacementof some amino acids within the transgene sequence. Fusions constructedfor chloroplast import can be tested for efficacy of chloroplast uptakeby in vitro translation of in vitro transcribed constructions followedby in vitro chloroplast uptake using techniques described, for example,by Bartlett et al., in: Edelmann et al., (eds.) Methods in ChloroplastMolecular Biology, Elsevier pp 1081-1091 (1982) and Wasmann et al.,(1986) Mol. Gen. Genet. 205: 446-453.

These construction techniques are well known in the art and are equallyapplicable to mitochondria and peroxisomes.

The above-described mechanisms for cellular targeting can be utilizednot only in conjunction with their cognate promoters, but also inconjunction with heterologous promoters so as to effect a specificcell-targeting goal under the transcriptional regulation of a promoterthat has an expression pattern different to that of the promoter fromwhich the targeting signal derives.

V. Construction of Plant Transformation Vectors

Numerous transformation vectors available for plant transformation areknown to those of ordinary skill in the plant transformation arts, andthe genes pertinent to this invention can be used in conjunction withany such vectors. The selection of vector will depend upon the preferredtransformation technique and the target species for transformation. Forcertain target species, different antibiotic or herbicide selectionmarkers can be preferred. Selection markers used routinely intransformation include the nptII gene, which confers resistance tokanamycin and related antibiotics (Messing & Vierra, (1982) Gene 19:259-268; Bevan et al., (1983) Nature 304:184-187), the bar gene, whichconfers resistance to the herbicide phosphinothricin (White et al.,(1990) Nucl. Acids Res 18: 1062, Spencer et al., (1990) Theor. Appl.Genet 79: 625-631), the hph gene, which confers resistance to theantibiotic hygromycin (Biochinger & Diggelmann, Mol Cell Biol 4:2929-2931), and the dhfr gene, which confers resistance to methatrexate(Bourouis et al., (1983) EMBO J. 2(7): 1099-1104), and the EPSPS gene,which confers resistance to glyphosate (U.S. Pat. Nos. 4,940,935 and5,188,642).

V.A. Transformation

Once the coding sequence of interest has been cloned into an expressionsystem, it can then be transformed into a plant cell. Methods fortransformation and regeneration of plants are well known in the art. Forexample, Ti plasmid vectors have been utilized for the delivery offoreign DNA, as well as direct DNA uptake, liposomes, electroporation,microinjection, and microprojectiles. In addition, bacteria from thegenus Agrobacterium can be utilized to transform plant cells. Below aredescriptions of some representative techniques for transforming bothdicotyledonous and monocotyledonous plants.

V.A.1. Transformation of Dicotyledons

Transformation techniques for dicotyledons are well known in the art andinclude Agrobacterium-based techniques and techniques that do notrequire Agrobacterium. Non-Agrobacterium techniques involve the uptakeof exogenous genetic material directly by protoplasts or cells. This canbe accomplished by PEG or electroporation mediated uptake, particlebombardment-mediated delivery, or microinjection. Examples of thesetechniques are described by Paszkowski et al., (1984) EMBO J 3:2717-2722, Potrykus et al., (1985) Mol. Gen. Genet. 199: 169-177, Reichet al., (1986) Biotechnology 4: 1001-1004, and Klein et al., (1987)Nature 327: 70-73. In each case the transformed cells are regenerated towhole plants using standard techniques known in the art.

Agrobacterium-mediated transformation is a preferred technique fortransformation of dicotyledons because of its high efficiency oftransformation and its broad utility with many different species.Agrobacterium transformation typically involves the transfer of thebinary vector carrying the foreign DNA of interest (e.g. pCIB200 orpCIB2001) to an appropriate Agrobacterium strain, which can depend ofthe complement of vir genes carried by the host Agrobacterium straineither on a co-resident Ti plasmid or chromosomally (e.g. strain CIB542for pCIB200 and pCIB2001 (Uknes et al., (1993) Plant Cell 5: 159-169).The transfer of the recombinant binary vector to Agrobacterium isaccomplished by a triparental mating procedure using E. coli carryingthe recombinant binary vector, a helper E. coli strain which carries aplasmid such as pRK2013 and which is able to mobilize the recombinantbinary vector to the target Agrobacterium strain. Alternatively, therecombinant binary vector can be transferred to Agrobacterium by DNAtransformation (Hofgen & Willmitzer, (1988) Nucl. Acids Res. 16: 9877).

Transformation of the target plant species by recombinant Agrobacteriumusually involves co-cultivation of the Agrobacterium with explants fromthe plant and follows protocols well known in the art. Transformedtissue is regenerated on selectable medium carrying the antibiotic orherbicide resistance marker present between the binary plasmid T-DNAborders.

Another approach to transforming plant cells with a gene involvespropelling inert or biologically active particles at plant tissues andcells. This technique is disclosed in U.S. Pat. Nos. 4,945,050,5,036,006, and 5,100,792, all to Sanford et al. Generally, thisprocedure involves propelling inert or biologically active particles atthe cells under conditions effective to penetrate the outer surface ofthe cell and afford incorporation within the interior thereof. Wheninert particles are utilized, the vector can be introduced into the cellby coating the particles with the vector containing the desired gene.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried yeast cells, dried bacteriumor a bacteriophage, each containing DNA sought to be introduced) canalso be propelled into plant cell tissue.

V.A.2. Transformation of Monocotyledons

Transformation of most monocotyledon species has now also becomeroutine. Representative techniques include direct gene transfer intoprotoplasts using PEG or electroporation techniques, and particlebombardment into callus tissue. Transformations can be undertaken with asingle DNA species or multiple DNA species (i.e. co-transformation) andboth these techniques are suitable for use with this invention.Co-transformation can have the advantage of avoiding complete vectorconstruction and of generating transgenic plants with unlinked loci forthe gene of interest and the selectable marker, enabling the removal ofthe selectable marker in subsequent generations, should this be regardeddesirable.

Patent Applications EP 0 292 435, EP 0 392 225, and WO 93/07278 describetechniques for the preparation of callus and protoplasts from an eliteinbred line of maize, transformation of protoplasts using PEG orelectroporation, and the regeneration of maize plants from transformedprotoplasts. Gordon-Kamm et al. (Gordon-Kamm et al., (1990) Plant Cell2: 603-618) and Fromm et al. (Fromm et al., (1990) Biotechnology 8:833-839) have published techniques for transformation of A188-derivedmaize line using particle bombardment. Furthermore, WO 93/07278 andKoziel et al. (Koziel et al., (1993) Biotechnology 11: 194-200) describetechniques for the transformation of elite inbred lines of maize byparticle bombardment. This technique utilizes immature maize embryos of1.5-2.5 mm length excised from a maize ear 14-15 days after pollinationand a PDS-1000 He Biolistics device for bombardment.

Transformation of rice can also be undertaken by direct gene transfertechniques utilizing protoplasts or particle bombardment.Protoplast-mediated transformation has been described for Japonica-typesand Indica-types (Zhang et al., (1988) Plant Cell Rep 7: 379-384;Shimamoto et al., (1989) Nature 338: 274-277; Datta et al., (1990)Biotechnology 8: 736-740). Both types are also routinely transformableusing particle bombardment (Christou et al., (1991) Biotechnology 9:957-962). Furthermore, WO 93/21335 describes techniques for thetransformation of rice via electroporation. Patent Application EP 0 332581 describes techniques for the generation, transformation andregeneration of Pooideae protoplasts. These techniques allow thetransformation of Dactylis and wheat. Furthermore, wheat transformationhas been described by Vasil et al. (Vasil et al., (1992) Biotechnology10: 667-674) using particle bombardment into cells of type C long-termregenerable callus, and also by Vasil et al. (Vasil et al., (1993)Biotechnology 11: 1553-1558) and Weeks et al. (Weeks et al., (1993)Plant Physiol. 102: 1077-1084) using particle bombardment of immatureembryos and immature embryo-derived callus. One technique for wheattransformation, however, involves the transformation of wheat byparticle bombardment of immature embryos and includes either a highsucrose or a high maltose step prior to gene delivery. Prior tobombardment, any number of embryos (0.75-1 mm in length) are plated ontoMS medium with 3% sucrose (Murashiga & Skoog, (1962) PhysiologiaPlantarum 15: 473-497) and 3 mg/l 2,4-D for induction of somaticembryos, which is allowed to proceed in the dark. On the chosen day ofbombardment, embryos are removed from the induction medium and placedonto the osmoticum (i.e. induction medium with sucrose or maltose addedat the desired concentration, typically 15%). The embryos are allowed toplasmolyze for 2-3 h and are then bombarded. Twenty embryos per targetplate is typical, although not critical.

An appropriate gene-carrying plasmid (such as pCIB3064 or pSG35) isprecipitated onto micrometer size gold particles using standardprocedures. Each plate of embryos is shot with the DuPont BIOLISTICS®helium device using a burst pressure of about 1000 psi using a standard80 mesh screen. After bombardment, the embryos are placed back into thedark to recover for about 24 h (still on osmoticum). After 24 hrs, theembryos are removed from the osmoticum and placed back onto inductionmedium where they stay for about a month before regeneration.Approximately one month later the embryo explants with developingembryogenic callus are transferred to regeneration medium (MS+1 mg/literNAA, 5 mg/liter GA), further containing the appropriate selection agent(10 mg/l basta in the case of pCIB3064 and 2 mg/l methotrexate in thecase of pSOG35). After approximately one month, developed shoots aretransferred to larger sterile containers known as “GA7s” which containhalf-strength MS, 2% sucrose, and the same concentration of selectionagent.

More recently, tranformation of monocotyledons using Agrobacterium hasbeen described. See WO 94/00977 and U.S. Pat. No. 5,591,616, both ofwhich are incorporated herein by reference.

VI. Transgenic Plants

A “transgenic plant” is one that has been genetically modified tocontain and express heterologous DNA sequences, either as regulatory RNAmolecules or as proteins. A transgenic plant can be genetically modifiedto contain and express at least one homologous or heterologous DNAsequence operably linked to and under the regulatory control oftranscriptional control sequences which function in plant cells ortissue or in whole plants. As used herein, a transgenic plant alsorefers to progeny of the initial transgenic plant where those progenycontain and are capable of expressing the homologous or heterologouscoding sequence under the regulatory control of the plant-expressibletranscription control sequences described herein. Seeds containingtransgenic embryos are encompassed within this definition as arecuttings and other plant materials for vegetative propagation of atransgenic plant.

When plant expression of a homologous or heterologous gene or codingsequence of interest is desired, that coding sequence is operably linkedin the sense orientation to a suitable promoter and advantageously underthe regulatory control of DNA sequences which quantitatively regulatetranscription of a downstream sequence in plant cells or tissue or inplanta, in the same orientation as the promoter, so that a sense (i.e.,functional for translational expression) mRNA is produced. Atranscription termination signal, for example, as polyadenylationsignal, functional in a plant cell is advantageously placed downstreamof an LOL1 coding sequence, and a selectable marker which can beexpressed in a plant, can be covalently linked to the inducibleexpression unit so that after this DNA molecule is introduced into aplant cell or tissue, its presence can be selected and plant cells ortissue not so transformed will be killed or prevented from growing.

Where tissue specific expression of the plant-expressible LOL1 codingsequence is desired, the skilled artisan will choose from a number ofwell-known sequences to mediate that form of gene expression asdisclosed herein. Environmentally regulated promoters are also wellknown in the art, and the skilled artisan can choose from well-knowntranscription regulatory sequences to achieve the desired result.

A method for providing positive regulation of cell death and/or adisease resistance characteristic to a plant is therefore disclosed. Themethod comprises introducing to said plant a construct comprising anucleic acid sequence encoding an LOL1 gene product operatively linkedto a promoter, wherein production of the LOL1 gene product in the plantprovides positive regulation of cell death and/or a disease resistancecharacteristic in the plant. The construct can further comprises avector selected from the group consisting of a plasmid vector or a viralvector. The LOL1 gene product comprises a protein having an amino acidsequence as set forth in any of SEQ ID NOs: 2 and 4-12. The nucleic acidsequence can be selected from the group including, but not limited to,(a) SEQ ID NO: 1; (b) a sequence encoding a polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NOs: 2and 4-12; and (c) a nucleic acid sequence capable of hybridizing understringent conditions to a nucleic acid sequence according to (a) or (b).

In an alternative embodiment, the construct further comprises anothernucleic acid molecule encoding a polypeptide that provides an additionaldesired characteristic to the plant. Other desired characteristicsinclude, for example, yield, drought resistance, chemical resistance(e.g. herbicide or pesticide resistance), spoilage resistance or any orother desired characteristic as would be apparent to one of ordinaryskill in the art after review of the disclosure of the presentinvention. Representative nucleic acids sequences are described in thefollowing U.S. patents: U.S. Pat. No. 5,948,953 to Webb (brown rotfungus resistance); U.S. Pat No. Re. 36,449 to Lebrun et al. (herbicideresistance); U.S. Pat. No. 5,952,546 to Bedbrook et al. (delayedripening tomato plants); and U.S. Pat. No. 5,986,173 issued Nov. 16,1999 to Smeekens et al. (transgenic plants showing a modified fructanpattern).

Optionally, the method further comprises monitoring an insertion pointfor the construct in the plant genome; and providing for insertion ofthe construct into the plant genome at a location not associated withthe resistance characteristic, the desired characteristic, or both theresistance or the desired characteristic.

Disease resistance and/or positive regulation of cell death can beconferred to a wide variety of plant cells, including those ofgymnosperms, monocots, and dicots. Although the gene can be insertedinto any plant cell falling within these broad classes, it can beparticularly useful in crop plant cells, such as rice, wheat, barley,rye, corn, potato, carrot, sweet potato, sugar beet, bean, pea, chicory,lettuce, cabbage, cauliflower, broccoli, turnip, radish, spinach,asparagus, onion, garlic, eggplant, pepper, celery, carrot, squash,pumpkin, zucchini, cucumber, apple, pear, quince, melon, plum, cherry,peach, nectarine, apricot, strawberry, grape, raspberry, blackberry,pineapple, avocado, papaya, mango, banana, tobacco, tomato, sorghum andsugarcane.

VII. Plant Breeding

A high-level expression of the LOL1 gene, and mutants thereof, can beincorporated into plant lines through breeding. Breeding approaches andtechniques are known in the art. See, for example, Welsh, Fundamentalsof Plant Genetics and Breeding, John Wiley & Sons, New York (1981); CropBreeding, Wood (Ed.), American Society of Agronomy, Madison, Wis.(1983); Mavo, The Theory of Plant Breeding, Second Edition, ClarendonPress, Oxford (1987); Singh, Breeding for Resistance to Diseases andInsect Pests, Springer-Verlag, New York (1986); and Wricke & Weber,Quantitative Genetics and Selection Plant Breeding, Walter de Gruyterand Co., Berlin (1986).

VIII. Design and Preparation of LOL1 Mutants

The present invention provides for the generation of LOL1 mutants. Ageneral discussion of the design and preparation of such mutants andstructural equivalents is presented hereinbelow. The following isdiscussion is also generally applicable to the LOL1 wild typepolypeptides of the present invention in various contexts, such as butnot limited to sequence identity, functional and biological equivalents,and sequence substitutions.

VIII.A. Chimeric LOL1 Polypeptides

The generation of chimeric LOL1 polypeptides is an aspect of the presentinvention. Such a chimeric polypeptide can comprise a LOL1 polypeptideor a portion of a LOL1, which is fused to a candidate polypeptide or asuitable region of the candidate polypeptide, for example a LOL1expressed in a species other than Arabidopsis. Throughout the presentdisclosure it is intended that the term “mutant” encompass not onlymutants of a LOL1 polypeptide but chimeric proteins generated using aLOL1 as well. Thus, it is intended that the following discussion ofmutant LOL1 polypeptides apply mutatis mutandis to chimeric LOL1polypeptides and to structural equivalents thereof.

In accordance with the present invention, a mutation can be directed toa particular site or combination of sites of a wild-type LOL1. Forexample, a residue having a location on, at or near the surface of thepolypeptide can be replaced, resulting in an altered surface charge ofone or more charge units, as compared to the wild-type LOL1.Alternatively, an amino acid residue in a LOL1 can be chosen forreplacement based on its hydrophilic or hydrophobic characteristics.

Such mutants can be characterized by any one of several differentproperties as compared with the wild-type LOL1. For example, suchmutants can have an altered surface charge of one or more charge units,or can have an increase in overall stability. Other mutants can havealtered substrate specificity in comparison with, or a higher specificactivity than, a wild-type LOL1.

LOL1 mutants of the present invention can be generated in a number ofways. For example, the wild-type sequence of a LOL1 can be mutated atthose sites identified using this invention as desirable for mutation,by oligonucleotide-directed mutagenesis or other conventional methods,such as deletion. Alternatively, mutants of a LOL1 can be generated bythe site-specific replacement of a particular amino acid with anunnaturally occurring amino acid. In addition, LOL1 mutants can begenerated through replacement of an amino acid residue, for example, aparticular cysteine or methionine residue, with selenocysteine orselenomethionine. This can be achieved by growing a host organismcapable of expressing either the wild-type or mutant polypeptide on agrowth medium depleted of either natural cysteine or methionine (orboth) but enriched in selenocysteine or selenomethionine (or both).

A mutation can be introduced into a DNA sequence coding for a LOL1 usingsynthetic oligonucleotides. These oligonucleotides contain nucleotidesequences flanking the desired mutation sites. A mutation can begenerated in the full-length DNA sequence of a LOL1 or in any sequencecoding for polypeptide fragments of a LOL1.

According to the present invention, a mutated LOL1 DNA sequence producedby the methods described above, or any alternative methods known in theart, can be expressed using an expression vector. An expression vector,as is well known to those of skill in the art, typically includeselements that permit autonomous replication in a host cell independentof the host genome, and one or more phenotypic markers for selectionpurposes. Either prior to or after insertion of the DNA sequencessurrounding the desired LOL1 mutant coding sequence, an expressionvector also will include control sequences encoding a promoter,operator, ribosome binding site, translation initiation signal, and,optionally, a repressor gene or various activator genes and a signal fortermination. In some embodiments, where secretion of the produced mutantis desired, nucleotides encoding a “signal sequence” can be insertedprior to a LOL1 mutant coding sequence. For expression under thedirection of the control sequences, a desired DNA sequence must beoperatively linked to the control sequences; that is, the sequence musthave an appropriate start signal in front of the DNA sequence encodingthe LOL1 mutant, and the correct reading frame to permit expression ofthat sequence under the control of the control sequences and productionof the desired product encoded by that LOL1 sequence must be maintained.

Any of a wide variety of well-known available expression vectors can beuseful in the expression of a mutated LOL1 coding sequence of thisinvention. These expression vectors can be used in the techniquesdisclosed herein above and in the Laboratory Examples and can include,for example, vectors comprising segments of chromosomal, non-chromosomaland synthetic DNA sequences, such as various known derivatives of SV40,known bacterial plasmids, e.g., plasmids from E. coli including col E1,pCR1, pBR322, pMB9 and their derivatives, wider host range plasmids,e.g., RP4, phage DNAs, e.g., the numerous derivatives of phage λ, e.g.,NM 989, and other DNA phages, e.g., M13 and filamentous single strandedDNA phages, yeast plasmids and vectors derived from combinations ofplasmids and phage DNAs, such as plasmids which have been modified toemploy phage DNA or other expression control sequences.

In addition, any of a wide variety of expression controlsequences—sequences that control the expression of a DNA sequence whenoperatively linked to it—can be used in these vectors to express themutated DNA sequences according to this invention. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40 for animal cells, the lac system, the trp system theTAC or TRC system, the major operator and promoter regions of phage λ,the control regions of fd coat protein, all for E. coli, the promoterfor 3-phosphoglycerate kinase or other glycolytic enzymes, the promotersof acid phosphatase, e.g., Pho5, the promoters of the yeast α-matingfactors for yeast, and other sequences known to control the expressionof genes of prokaryotic or eukaryotic cells or their viruses, andvarious combinations thereof.

A wide variety of hosts are also useful for producing mutated LOL1polypeptides according to this invention. These hosts include, forexample, bacteria, such as E. coli, Bacillus and Streptomyces, fungi,such as yeasts, plant cells, insect cells, such as Sf9 and Sf21 cells,and transgenic host cells.

It should be understood that not all expression vectors and expressionsystems function in the same way to express mutated DNA sequences ofthis invention, and to produce modified LOL1 polypeptides or LOL1mutants. Neither do all hosts function equally well with the sameexpression system. One of ordinary skill in the art can, however, make aselection among these vectors, expression control sequences and hostswithout undue experimentation and without departing from the scope ofthis invention. For example, an important consideration in selecting avector will be the ability of the vector to replicate in a given host.The copy number of the vector, the ability to control that copy number,and the expression of any other proteins encoded by the vector, such asantibiotic markers, should also be considered.

When selecting an expression control sequence, a variety of factorsshould also be considered. These include, for example, the relativestrength of the system, its controllability and its compatibility withthe DNA sequence encoding a modified LOL1 polypeptide of this invention,with particular regard to the formation of potential secondary andtertiary structures.

Hosts should be selected by consideration of their compatibility withthe chosen vector, the toxicity of a modified LOL1 to them, theirability to express mature products, their ability to fold proteinscorrectly, their fermentation requirements, the ease of purification ofa modified LOL1 and safety. Within these parameters, one of skill in theart can select various vector/expression control system/hostcombinations that will produce useful amounts of a mutant LOL1. A mutantLOL1 produced in these systems can be purified by a variety ofconventional steps and strategies, including those used to purify thewild-type LOL1.

Once a LOL1 mutation(s) has been generated in the desired location, suchas a ligand binding site, the mutants can be tested for any one ofseveral properties of interest. For example, mutants can be screened foran altered charge at physiological pH. This can be determined bymeasuring the mutant LOL1 isoelectric point (pI) and comparing theobserved value with that of the wild-type parent. Isoelectric point canbe measured by gel-electrophoresis according to the method of Wellner(Wellner, (1971) Anal. Chem. 43: 597). A mutant LOL1 polypeptidecontaining a replacement amino acid located at the surface of theenzyme, as provided by the structural information of this invention, canlead to an altered surface charge and an altered pI.

VIII.B. Generation of an Engineered LOL1 or LOL1 Mutant

In another aspect of the present invention, a unique LOL1 polypeptidecan be generated. Such a mutant can facilitate purification and/or canfacilitate the study of the biological activity of a LOL1 polypeptide.

As used herein, the terms “engineered LOL1” and “LOL1 mutant” refer topolypeptides having amino acid sequences that contain at least onemutation in the wild-type sequence. The terms also refer to LOL1polypeptides which are capable of exerting a biological effect in thatthey comprise all or a part of the amino acid sequence of an LOL1 mutantpolypeptide of the present invention, or cross-react with antibodiesraised against a LOL1 mutant polypeptide, or retain all or some or anenhanced degree of the biological activity of the LOL1 mutant amino acidsequence or protein. Such biological activity can include diseaseresistance and/or postive regulation of cell death.

The terms “engineered LOL1” and “LOL1 mutant” also includes analogs of aLOL1 mutant polypeptide. By “analog” is intended that a DNA orpolypeptide sequence can contain alterations relative to the sequencesdisclosed herein, yet retain all or some or an enhanced degree of thebiological activity of those sequences. Analogs can be derived fromgenomic nucleotide sequences or from other organisms, or can be createdsynthetically. Those of skill in the art will appreciate that otheranalogs, as yet undisclosed or undiscovered, can be used to designand/or construct LOL1 mutant analogs. There is no need for a LOL1 mutantpolypeptide to comprise all or substantially all of the amino acidsequence of SEQ ID NOs: 2 and 4-12. Shorter or longer sequences areanticipated to be of use in the invention; shorter sequences are hereinreferred to as “segments”. Thus, the terms “engineered LOL1” and “LOL1mutant” also includes fusion, chimeric or recombinant LOL1 or LOL1mutant polypeptides and proteins comprising sequences of the presentinvention. Methods of preparing such proteins are disclosed herein aboveand are known in the art.

VlII.C. Sequence Similarity and Identity

As used herein, the term “substantially similar” means that a particularsequence varies from nucleic acid sequence of SEQ ID NO: 1 or the aminoacid sequence of SEQ ID NOs: 2 and 4-12 by one or more deletions,substitutions, or additions, the net effect of which is to retain atleast some of biological activity of the natural gene, gene product, orsequence. Such sequences include “mutant” or “polymorphic” sequences, orsequences in which the biological activity and/or the physicalproperties are altered to some degree but retains at least some or anenhanced degree of the original biological activity and/or physicalproperties. In determining nucleic acid sequences, all subject nucleicacid sequences capable of encoding substantially similar amino acidsequences are considered to be substantially similar to a referencenucleic acid sequence, regardless of differences in codon sequences orsubstitution of equivalent amino acids to create biologically functionalequivalents.

VIII.C.1. Sequences that are Substantially Identical to a LOL1 Sequence

Nucleic acids that are substantially identical to a nucleic acidsequence of a LOL1 sequence of the present invention (including a LOL1mutant), such as allelic variants, genetically altered versions of thegene, etc., bind to a LOL1 sequence under stringent hybridizationconditions. By using probes, particularly labeled probes of DNAsequences, one can isolate homologous or related genes. The source ofhomologous genes can be any species.

Between various plant species, homologs can have substantial sequencesimilarity, i.e. at least 85%-99% sequence identity between nucleotidesequences, including at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, and 98% sequence identity. Sequence similarity iscalculated based on a reference sequence, which can be a subset of alarger sequence, such as a conserved motif, coding region, flankingregion, etc. A reference sequence can be, for example, at least about 18nucleotides (nt) long, or in another example, at least about 30nucleotides long, and can extend to the complete sequence that is beingcompared. Algorithms for sequence analysis are known in the art, such asBLAST, described in Altschul et al., (1990) J. Mol. Biol. 215: 403-10.

Percent identity or percent similarity of a DNA or peptide sequence canbe determined, for example, by comparing sequence information using theGAP computer program, available from the University of WisconsinGeneticist Computer Group. The GAP program utilizes the alignment methodof Needleman et al., (1970) J. Mol. Bio. 48: 443, as revised by Smith etal., (1981) Adv. Appl. Math. 2:482. Briefly, the GAP program definessimilarity as the number of aligned symbols (i.e., nucleotides or aminoacids) that are similar, divided by the total number of symbols in theshorter of the two sequences. Parameters for the GAP program can be, forexample, the default parameters, which do not impose a penalty for endgaps. See, e.g., Schwartz et al., (eds.), (1979), Atlas of ProteinSequence and Structure, National Biomedical Research Foundation, pp.357-358, and Gribskov et al., (1986) Nucl. Acids. Res. 14: 6745.

The term “similarity” is contrasted with the term “identity”. Similarityis defined as above; “identity”, however, means a nucleic acid or aminoacid sequence having the same amino acid at the same relative positionin a given family member of a gene family. Homology and similarity aregenerally viewed as broader terms than the term identity. Biochemicallysimilar amino acids, for example leucine/isoleucine orglutamate/aspartate, can be present at the same position—these are notidentical per se, but are biochemically “similar.” As disclosed herein,these are referred to as conservative differences or conservativesubstitutions. This differs from a conservative mutation at the DNAlevel, which changes the nucleotide sequence without making a change inthe encoded amino acid, e.g. TCC to TCA, both of which encode serine.

As used herein, nucleic acid sequences are “substantially identical” tospecific nucleic acid disclosed herein if: (a) the nucleic acid sequenceis derived from coding regions of the nucleic acid sequence shown in SEQID NO: 1; or (b) the nucleic acid sequence is capable of hybridizationwith nucleic acid sequences of (a) under stringent conditions and whichencode a biologically active LOL1 gene product; or (c) the nucleic acidsequences are degenerate as a result of alternative genetic code to thenucleic acid sequences defined in (a) and/or (b). Substantiallyidentical proteins and nucleic acids can have, for example, betweenabout 70% and 80%, or about 81% to about 90% or about 91% and 99%sequence identity with the corresponding sequence of the native proteinor nucleic acid. Sequences having lesser degrees of identity butcomparable biological activity are considered to be equivalents.

As used herein, “stringent conditions” means conditions of highstringency, for example 6×SSC, 0.2% polyvinylpyrrolidone, 0.2% Ficoll,0.2% bovine serum albumin, 0.1% sodium dodecyl sulfate, 100 μg/ml salmonsperm DNA and 15% formamide at 68° C. For the purposes of specifyingadditional conditions of high stringency, conditions can comprise, forexample, a salt concentration of about 200 mM and temperature of about45° C. One example of such stringent conditions is hybridization at4×SSC, at 65° C., followed by a washing in 0.1×SSC at 65° C. for onehour. Another example stringent hybridization scheme uses 50% formamide,4×SSC at 42° C.

In contrast, nucleic acids having sequence similarity are detected byhybridization under lower stringency conditions. Thus, sequence identitycan be determined by hybridization under lower stringency conditions,for example, at 50° C. or higher and 0.1×SSC (9 mM NaCl/0.9 mM sodiumcitrate) and the sequences will remain bound when subjected to washingat 55° C. in 1×SSC.

VIII.C.2. Complementarity and Hybridization to a LOL1 Sequence

As used herein, the term “complementary sequences” means nucleic acidsequences that are base-paired according to the standard Watson-Crickcomplementarity rules. The present invention also encompasses the use ofnucleotide segments that are complementary to the sequences of thepresent invention.

Hybridization can also be used for assessing complementary sequencesand/or isolating complementary nucleotide sequences. As discussed above,nucleic acid hybridization will be affected by such conditions as saltconcentration, temperature, or organic solvents, in addition to the basecomposition, length of the complementary strands, and the number ofnucleotide base mismatches between the hybridizing nucleic acids, aswill be readily appreciated by those skilled in the art. Stringenttemperature conditions will generally include temperatures in excess ofabout 30° C., typically in excess of about 37° C., and or temperaturesin excess of about 45° C. Stringent salt conditions will ordinarily beless than about 1,000 mM, less than about 500 mM, or less than about 200mM. However, the combination of parameters is much more important thanthe measure of any single parameter. See, e.g., Wetmur & Davidson,(1968) J. Mol. Biol. 31: 349-70. Determining appropriate hybridizationconditions to identify and/or isolate sequences containing high levelsof homology is well known in the art. See, e.g., Sambrook et al., (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York.

VIII.D. Functional Equivalents of a LOL1 Nucleic Acid Sequence of thePresent Invention

As used herein, the term “functionally equivalent codon” is used torefer to codons that encode the same amino acid, such as the ACG and AGUcodons for serine. LOL1-encoding nucleic acid sequences comprising SEQID NO: 1, and fragments thereof, which have functionally equivalentcodons, are covered by the present invention. Thus, when referring tothe sequence example presented in SEQ ID NO: 1, applicants contemplatesubstitution of functionally equivalent codons into the sequence exampleof SEQ ID NO: 1. Thus, applicants are in possession of amino acid andnucleic acids sequences which include such substitutions but which arenot set forth herein in their entirety for convenience.

It will also be understood by those of skill in the art that amino acidand nucleic acid sequences can include additional residues, such asadditional N— or C-terminal amino acids or 5′ or 3′ nucleic acidsequences, and yet still be essentially as set forth in one of thesequences disclosed herein, so long as the sequence retains biologicalprotein activity where polypeptide expression is concerned. The additionof terminal sequences particularly applies to nucleic acid sequenceswhich can, for example, include various non-coding sequences flankingeither of the 5′ or 3′ portions of the coding region or can includevarious internal sequences, i.e., introns, which are known to occurwithin genes.

VIII.E. Biological Equivalents

The present invention envisions and includes biological equivalents of aLOLL polypeptide of the present invention. The term “biologicalequivalent” refers to proteins having amino acid sequences which aresubstantially identical to the amino acid sequence of a LOL1 polypeptideof the present invention and which are capable of exerting a biologicaleffect in that they are capable of modulating cell death orcross-reacting with anti-LOL1 antibodies raised against a LOL1polypeptide (such as a mutant LOL1 polypeptide) of the presentinvention.

For example, certain amino acids can be substituted for other aminoacids in a protein structure without appreciable loss of interactivecapacity with, for example, structures in the nucleus of a cell. Sinceit is the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence (or the nucleic acidsequence encoding it) to obtain a protein with the same, enhanced, orantagonistic properties. Such properties can be achieved by interactionwith the normal targets of the protein, but this need not be the case,and the biological activity of the invention is not limited to aparticular mechanism of action. It is thus in accordance with thepresent invention that various changes can be made in the amino acidsequence of a LOL1 polypeptide of the present invention (including aLOL1 mutant) or its underlying nucleic acid sequence without appreciableloss of biological utility or activity.

Biologically equivalent polypeptides, as used herein, are polypeptidesin which certain, but not most or all, of the amino acids can besubstituted. Thus, when referring to the sequence examples presented inSEQ ID NOs: 2 and 4-12 applicants envision substitution of codons thatencode biologically equivalent amino acids, as described herein, intothe sequence examples of SEQ ID NOs: 2 and 4-12, respectively. Thus,applicants are in possession of amino acid and nucleic acids sequenceswhich include such substitutions but which are not set forth herein intheir entirety for convenience.

Alternatively, functionally equivalent proteins or peptides can becreated via the application of recombinant DNA technology, in whichchanges in the protein structure can be engineered, based onconsiderations of the properties of the amino acids being exchanged,e.g. substitution of lIe for Leu. Changes designed by man can beintroduced through the application of site-directed mutagenesistechniques, e.g., to introduce improvements to the antigenicity of theprotein or to test a mutant LOL1 polypeptide of the present invention inorder to modulate cell death or other activity, at the molecular level.

Amino acid substitutions, such as those which might be employed inmodifying a LOL1 polypeptide of the present invention are generally, butnot necessarily, based on the relative similarity of the amino acidside-chain substituents, for example, their hydrophobicity,hydrophilicity, charge, size, and the like. An analysis of the size,shape and type of the amino acid side-chain substituents reveals thatarginine, lysine and histidine are all positively charged residues; thatalanine, glycine and serine are all of similar size; and thatphenylalanine, tryptophan and tyrosine all have a generally similarshape. Therefore, based upon these considerations, arginine, lysine andhistidine; alanine, glycine and serine; and phenylalanine, tryptophanand tyrosine; are defined herein as biologically functional equivalents.Other biologically functionally equivalent changes will be appreciatedby those of ordinary skill in the art. It is implicit in the abovediscussion, however, that one of skill in the art can appreciate that aradical, rather than a conservative substitution is warranted in a givensituation. Non-conservative substitutions in LOL1 polypeptides(including LOL1 mutant polypeptides) of the present invention are alsoan aspect of the present invention.

In making biologically functional equivalent amino acid substitutions,the hydropathic index of amino acids can be considered. Each amino acidhas been assigned a hydropathic index on the basis of theirhydrophobicity and charge characteristics, these are: isoleucine (+4.5);valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine (+2.5);methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7);serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6);histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5);asparagine (−3.5); lysine (−3.9); and arginine (−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is generally understood inthe art (Kyte & Doolittle, (1982), J. Mol. Biol. 157: 105-132). It isknown that certain amino acids can be substituted for other amino acidshaving a similar hydropathic index or score and still retain a similarbiological activity. In making changes based upon the hydropathic index,the substitution of amino acids whose hydropathic indices are within,for example, ±2, ±1, or ±0.5 of the original value can also be employed.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, incorporated herein by reference, states that thegreatest local average hydrophilicity of a protein, as governed by thehydrophilicity of its adjacent amino acids, correlates with itsimmunogenicity and antigenicity, i.e. with a biological property of theprotein. It is understood that an amino acid can be substituted foranother having a similar hydrophilicity value and still obtain abiologically equivalent protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3);asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4);proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0);methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8);tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

In making changes based upon similar hydrophilicity values, thesubstitution of amino acids whose hydrophilicity values are within, forexample, ±2, ±1 or within ±0.5 of the original value can be employed.

While discussion has focused on functionally equivalent polypeptidesarising from amino acid changes, it will be appreciated that thesechanges can be effected by alteration of the encoding DNA, taking intoconsideration also that the genetic code is degenerate and that two ormore codons can code for the same amino acid.

Thus, it will also be understood that this invention is not limited tothe particular amino acid and nucleic acid sequences of SEQ ID NOs: 1and 4-12. Recombinant vectors and isolated DNA segments can thereforevariously include a LOL1 polypeptide-encoding region (including a mutantLOL1 polypeptide-encoding region) itself, include coding regions bearingselected alterations or modifications in the basic coding region, orinclude larger polypeptides which nevertheless comprise a LOL1polypeptide-encoding region (including a mutant LOL1polypeptide-encoding region) or can encode biologically functionalequivalent proteins or polypeptides which have variant amino acidsequences. Biological activity of a LOL1 polypeptide can be determined,for example, by assay disclosed herein.

The nucleic acid segments of the present invention, regardless of thelength of the coding sequence itself, can be combined with other DNAsequences, such as promoters, enhancers, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length can varyconsiderably. It is therefore contemplated that a nucleic acid fragmentof almost any length can be employed, with the total length being areflection of, for example, the ease of preparation and use in theintended recombinant DNA protocol. For example, nucleic acid fragmentscan be prepared which include a short stretch complementary to a nucleicacid sequence set forth in SEQ ID NO: 1, such as about 10 nucleotides,and which are up to 10,000 or 5,000 base pairs in length. DNA segmentswith total lengths of about 4,000, 3,000, 2,000, 1,000, 500, 200, 100,and about 50 base pairs in length can also be employed.

The DNA segments of the present invention encompass biologicallyfunctional equivalents of LOL1 polypeptides. Such sequences can rise asa consequence of codon redundancy and functional equivalency that areknown to occur naturally within nucleic acid sequences and the proteinsthus encoded. Alternatively, functionally equivalent proteins orpolypeptides can be created via the application of recombinant DNAtechnology, in which changes in the protein structure can be engineered,based on considerations of the properties of the amino acids beingexchanged. Changes can be introduced through the application ofsite-directed mutagenesis techniques, e.g., to introduce improvements tothe antigenicity of the protein or to test variants of a LOL1 of thepresent invention in a desired activity at the molecular level. Varioussite-directed mutagenesis techniques are known to those of ordinaryskill in the art and can be employed in the present invention.

The invention further encompasses fusion proteins and peptides wherein awild type or a mutant LOL1 coding region of the present invention isaligned within the same expression unit with other proteins or peptideshaving desired functions, such as for purification or immunodetectionpurposes.

Recombinant vectors form important further aspects of the presentinvention. Particularly useful vectors are those in which the codingportion of the DNA segment is positioned under the control of apromoter. The promoter can be that naturally associated with a LOL1gene, as can be obtained by isolating the 5′ non-coding sequenceslocated upstream of the coding segment or exon, for example, usingrecombinant cloning and/or PCR technology and/or other methods known inthe art, in conjunction with the compositions disclosed herein.

As disclosed herein above, in other embodiments, certain advantages willbe gained by positioning the coding DNA segment under the control of arecombinant, or heterologous, promoter. As used herein, a recombinant orheterologous promoter is a promoter that is not normally associated witha LOL1 gene in its natural environment. Such promoters can includepromoters isolated from bacterial, viral, eukaryotic, or plant cells.Naturally, it will be important to employ a promoter that effectivelydirects the expression of the DNA segment in the cell type chosen forexpression. The use of promoter and cell type combinations for proteinexpression is disclosed herein above and is generally known to those ofskill in the art of molecular biology (see, e.g., Sambrook et al.,(1989) Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York, incorporated herein by reference). As discussedabove, the promoters employed can be constitutive or inducible and canbe used under the appropriate conditions to direct high level expressionof the introduced DNA segment, such as is advantageous in thelarge-scale production of recombinant proteins or peptides.

IX. Methods, Compositions, and Kits for Detecting a LOL1 Polypeptide ora Nucleic Acid Molecule Encoding the Same

In another aspect of the invention, a method is provided for detecting aLOL1 polypeptide using an antibody that specifically recognizes a LOL1polypeptide, or portion thereof. In a preferred embodiment, biologicalsamples from an experimental subject and a control subject are obtained,and LOL1 polypeptide is detected in each sample by immunochemicalreaction with the antibody. More preferably, the antibody recognizesamino acids of any one of SEQ ID NOs:2 and 4-12, and is preparedaccording to a method of the present invention for producing such anantibody. A kit for carrying out the method is also provided.

In one embodiment, an antibody is used to screen a biological sample forthe presence of a LOL1 polypeptide. A biological sample to be screenedcan be a biological fluid such as extracellular or intracellular fluid,or a cell or tissue extract or homogenate. A biological sample can alsobe an isolated cell (e.g., in culture) or a collection of cells such asin a tissue sample or histology sample. A tissue sample can be suspendedin a liquid medium or fixed onto a solid support such as a microscopeslide. In accordance with a screening assay method, a biological sampleis exposed to an antibody immunoreactive with a LOL1 polypeptide whosepresence is being assayed, and the formation of antibody-polypeptidecomplexes is detected. Techniques for detecting such antibody-antigenconjugates or complexes are well known in the art and include but arenot limited to centrifugation, affinity chromatography and the like, andbinding of a labeled secondary antibody to the antibody-candidatereceptor complex.

The term “immunochemical reaction”, as used herein, refers to any of avariety of immunoassay formats used to detect antibodies specificallybound to a particular protein, including but not limited to competitiveand non-competitive assay systems using techniques such asradioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitationreactions, immunodiffusion assays, in situ immunoassays (e.g., usingcolloidal gold, enzyme or radioisotope labels), western blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc. See Harlow & Lane (1988) for a description of immunoassayformats and conditions.

The present invention also provides antibodies immunoreactive with aLOLL polypeptide. The term “antibody” indicates an immunoglobulinprotein, or functional portion thereof, including a polyclonal antibody,a monoclonal antibody, a chimeric antibody, a single chain antibody, Fabfragments, and an Fab expression library.

The term “functional portion” refers to the part of the protein thatbinds a molecule of interest. In a preferred embodiment, an antibody ofthe invention is a monoclonal antibody. Techniques for preparing andcharacterizing antibodies are well known in the art (See, e.g., Harlow &Lane, (1988) Antibodies: A Laboratory Manual Cold Spring HarborLaboratory Press, Cold Spring Harbor, New York). A monoclonal antibodyof the present invention can be readily prepared through use ofwell-known techniques such as the hybridoma techniques exemplified inU.S. Pat. No 4,196,265 and the phage-displayed techniques disclosed inU.S. Pat. No. 5,260,203.

The phrase “specifically (or selectively) binds to an antibody”, or“specifically (or selectively) immunoreactive with”, when referring to aprotein or peptide, refers to a binding reaction which is determinativeof the presence of the protein in a heterogeneous population of proteinsand other biological materials. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular protein and donot show significant binding to other proteins present in the sample.Specific binding to an antibody under such conditions can require anantibody that is selected for its specificity for a particular protein.For example, antibodies raised to a protein with an amino acid sequenceencoded by any of the nucleic acid sequences of the invention can beselected to obtain antibodies specifically immunoreactive with thatprotein and not with unrelated proteins.

The use of a molecular cloning approach to generate antibodies,particularly monoclonal antibodies, and more particularly single chainmonoclonal antibodies, are also provided. The production of single chainantibodies has been described in the art. See, e.g., U.S. Pat. No.5,260,203. For this approach, combinatorial immunoglobulin phagemidlibraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning on endothelial tissue. The advantages of thisapproach over conventional hybridoma techniques are that approximately10⁴ times as many antibodies can be produced and screened in a singleround, and that new specificities are generated by heavy (H) and light(L) chain combinations in a single chain, which further increases thechance of finding appropriate antibodies. Thus, an antibody of thepresent invention, or a “derivative” of an antibody of the presentinvention, pertains to a single polypeptide chain binding molecule whichhas binding specificity and affinity substantially similar to thebinding specificity and affinity of the light and heavy chain aggregatevariable region of an antibody described herein.

In another aspect of the invention, a method is provided for detecting anucleic acid molecule that encodes a LOL1 polypeptide. According to themethod, a biological sample having nucleic acid material is procured andhybridized under stringent hybridization conditions to a LOL1polypeptide-encoding nucleic acid molecule of the present invention.Such hybridization enables a nucleic acid molecule of the biologicalsample and a LOL1 polypeptide encoding-nucleic acid molecule to form adetectable duplex structure. Preferably, the LOL1 polypeptideencoding-nucleic acid molecule includes some or all nucleotides of SEQID NO:1.

An assay kit for detecting the presence of a LOL1 polypeptide that actsas a positive regulator of programmed cell death in a plant is alsodisclosed. In one embodiment, the assay kit comprises a first containercontaining a nucleic acid probe comprising a sequence of ten or morecontiguous nucleotide bases corresponding to a fragment of a nucleicacid sequence as disclosed herein. Optionally, the kit further comprisesa second container containing a detectable moiety, such as a radioactiveor fluorescent moiety, as would be apparent to one of ordinary skill inthe art after a review of the present disclosure.

LABORATORY EXAMPLES

The following Examples have been included to illustrate representativemodes of the invention. Certain aspects of, the following Examples aredescribed in terms of techniques and procedures found or contemplated bythe present inventors to work well in the practice of the invention.These Examples are exemplified through the use of standard laboratorypractices of the inventors. In light of the present disclosure and thegeneral level of skill in the art, those of skill will appreciate thatthe following Examples are intended to be exemplary only and thatnumerous changes, modifications and alterations can be employed withoutdeparting from the spirit and scope of the invention.

Standard recombinant DNA and molecular cloning techniques used here arewell known in the art and are described by Sambrook et al., (eds.)(1989) Molecular Cloning, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, New York and by Silhavy et al., (1984) Experiments withGene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NewYork and by Ausubel et al., (1992) Current Protocols in MolecularBiology John Wylie and Sons, Inc. New York.

Laboratory Example 1 Identification and Characterization of anLSD1-Related Gene, LOL1

In order to identify proteins with functions similar to that of LSD1, acomputer search was performed of the complete Arabidopsis genome usingthe internally conserved zinc finger motif sequence of LSD1 as listed inFIG. 2A: 5′-LXCXXCRXXLMYXXGASXVXCXXCXXXXXV (SEQ ID NO: 5). A smallLSD1-like gene family was identified. One of these genes, LOL1, mappedto BAC clone T9G5 at the top of chromosome 1 and encodes a protein of154 amino acids containing three LSD1-like zinc fingers (See FIG. 1).Another computer-aided homology search indicated that the nucleotidesequence of LOL1 is extremely highly conserved among monocotyledonousand dicotyledonous plants (FIG. 2B), but is absent from bacteria, yeastand animals. Like LSD1, LOL1 is constitutively expressed in all tissues.Its expression is unaltered in lsd1 null mutant plants grown underpermissive conditions.

Laboratory Example 2 Analysis of LOL1 Transcription in Wild-Type andMutant Plants

A T-DNA insertion mutant allele, lol1-1, was identified in which LOL1transcripts can no longer be detected by mRNA blot analysis. The lol1-1mutant was crossed to lsd1 and double mutants were identified using thepolymerase chain reaction (PCR). Different primer sets were used todetect the lsd1 mutation and the T-DNA insertion. To detect the lsd1mutation, the primers were 5′ACCTAACAAAAAGAAAAGTGTGTGAGG-3′ (SEQ ID NO:13), 5′-ATAATAACCCCTACTAGCTCTAACAAG-3′ (SEQ ID NO: 14), and5′-CTGCTACTTTCATCCAAAC-3′ (SEQ ID NO: 15). To detect the T-DNAinsertion, the primers were 5′-TGAGTTATGAGCAATATAGAGGAA-3′ (SEQ ID NO:16) and 5′-CATTTTATAATAACGCTGCGGACATCTAC-3′ (SEQ ID NO: 17).

Laboratory Example 3 Generation of Transgenic Arabidopsis ExpressingDifferent Levels of LOL1

Transgenic Arabidopsis lines were generated that expressed either higheror lower levels than wild-type levels of LOL1 mRNA (LOL1-s and lol1-as,respectively). Transgenic lines were established in both the wild typeWs-0 and isogenic lsd1 null backgrounds according to the method ofMcDowell et al., 1998. Briefly, the LOL1 coding region was cloned insense and antisense orientations into the binary vector pBAR1. LSD1/lsd1heterozygotes were transformed using standard techniques, and at least 6independent lines were identified per construct in the isogenic lsd1 andWs-0 backgrounds. All experiments described in the Laboratory Exampleswere carried out with at least four independent lines per construct pergenetic background.

RT-PCR was used to determine the relative mRNA levels of LOL1 in thevarious antisense transgenic lines using primers specific for the LOL1gene. These primers were 5′-CGAAACGCGATTCTACAATTAGTC-3′ (SEQ ID NO: 18)and 5′-ATTCACTCCAAGAAGAATTGCAAT-3′ (SEQ ID NO: 19). The nucleotide mixwith which the PCR reaction was performed contained a small amount ofα-³²P dATP, which allowed the products of the PCR reaction to beanalyzed using a phosphoimager device (such as are available fromAmersham Biosciences of Sunnyvale, Calif., United States of America)after they were separated on a 1.5% agarose gel. For the sense lines,RNA was analyzed with standard Northern blotting and hybridizationmethods using the LOL1 coding region as a radiolabelled probe, followedby analysis using a phosphoimager device. These techniques demonstratedthat LOL1 mRNA levels in the lol1-as lines were reduced to about 25-60%of wild type, and over-expression in LOL1-s lines resulted in ˜2-3 foldincrease in LOL1 transcript amount.

Laboratory Example 4 LOL1 Function is Required for lsd1 Rcd

In order to address whether manipulation of LOL1 levels altered eitherthe rcd induced in an lsd1 background or the wild type response topathogens, plants were treated withbenzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH). BTHinduces rcd in lsd1 plants (Görlach et al., (1996) Plant Cell8:629-643). Briefly, four-week-old plants from the lsd1/lol1-as andlsd1/LOL1-s lines were sprayed with 300 μM BTH and monitored for rcd. By7 days post-BTH spray, rcd devastated lsd1 and lsd1/LOL1-s leaves; thetissue was collapsed and completely dried. In contrast, lsd1/lol1-aslines were predominantly healthy and green. See FIG. 3A.

Cell death was examined by measuring conductivity as an indicator ofmembrane damage and cellular ion leakage as described in Baker et al.(Baker et al., (1993) Physiol. Molec. Plant Pathol. 43: 81-94) andDellagi et al. (Dellagi et al., (1998) Mol. Plant-Microbe Interact. 11:734-742). Briefly, at various stages after treatment, a 7 mm diameterleaf disk was removed using a cork borer, floated in distilled water for45 minutes, and transferred to tubes containing 6 ml of distilled water.Conductivity was determined with an Orion conductivity meter (availablefrom Thermo Orion of Beverly, Massachusetts, United States of America)at various time points. Mean and standard error were calculated from 4disks per genotype, with 3 repetitions within an experiment, and eachexperiment repeated 4 times. Ws-0 tissue did not exhibit any significantcell death and thus no increase in conductivity was observed, while lsd1mutant tissue reached a maximum conductivity at 96 hours after BTHtreatment. Reduction of LOL1 transcript levels in the lsd1/lol1-aslines, or in lsd1/lol1-1 significantly reduced conductivity compared toeither lsd1 or the lsd1/LOL1-s lines. See FIG. 3B.

Laboratory Example 5 Infection of Transgenic Arabidopsis with Botrytiscinerea

In order to assess the characteristics of rod in transgenic Arabidopsisplants that expressed different levels of LOL1, plants were infectedwith the necrotrophic pathogen Botrytis cinerea to activate rod. Thelsd1 rod phenotype is accelerated following B. cinerea infection,presumably because this fungus produces Reactive Oxygen Intermediates(ROI) as part of its pathogenicity program (Govrin & Levine, (2000)Curr. Biol. 10: 751-757). 4-week-old plants were drop-inoculated with B.cinerea and lsd1 rcd was visualized by lactophenol trypan blue stainingto identify dead cells (Keogh et al., (1980) Trans. Br. Mycol. Soc. 74:329-333). The particular isolate of B. cinerea used is weakly pathogenicon wild-type (i.e., LSD1-positive) Arabidopsis, in which staining islimited to the site of infection. In contrast, lsd1 leaves were killedby rcd and fungal proliferation. The lsd1/lol-as lines, on the otherhand, exhibited significantly reduced rod, while the lsd1/LOL-s lineswere at least as susceptible as lsd1. Lesion size was measured onseveral leaves per genotype. Reduction of LOL1 function clearlyattenuated rod in the lsd1 background, whereas over-expression of LOL1mRNA moderately enhanced rcd. See FIGS. 4A and 4B.

Laboratory Example 6 LOL1 is an Enhancer of HR

The LOL1 function observed in the aforementioned Examples was revealedin the context of an already poised ectopic cell death phenotype,namely, an lsd1 mutant genotype. In order to determine whethermis-regulation of LOL1 could also influence HR in a wild typebackground, Pst DC3000(avrRpm1) was used to trigger HR through theaction of the RPM1 disease resistance gene (Grant et al., (1995) Science269: 843-846) in the Ws-0, LOL1-s and lol1-as backgrounds. Briefly,4-week-old plants were infiltrated with a 10 mM MgCl₂ solutioncontaining Pst DC3000(avrRpm1) at a concentration of 5×10⁷ cfu/ml.Immediately thereafter, leaf disks were removed and processed as inExample 4. The onset of HR was quantified using conductivitymeasurements as in Example 4. The onset of HR using this assay was at 2hours post-inoculation (hpi), and maximum conductivity was reached at 6hpi. Thus, this assay correlated with the observed onset ofRPM1-dependent HR where tissue collapse is visible at ˜3 hpi and fulltissue collapse is evident by 6 hpi as reported by Dangl et al. (Danqlet al., (1992) Plant Cell 4: 1359-1369). The LOL1-s lines reached themaximum wild type conductivity level by 3.5 hpi, and achieved plateaulevels 20% higher than wild type. In this assay, therefore, the timecourse of cell collapse was accelerated in the LOL1-s lines.RPM1-mediated HR in the lol1-as lines was essentially wild type. SeeFIG. 5.

Laboratory Example 7 LOL1 Over-Expression Leads to Enhanced PathogenResistance

Since LOL1 has been shown to influence rcd, LOL-1s lines were tested forenhanced resistance to virulent pathogens by virtue of enhanced HR.4-week-old plants were sprayed with the virulent P. parasitica isolateEmco5 at 1×10⁴ spores/ml. Inoculated leaves were stained withlactophenol trypan blue at 5 days post inoculation (dpi). Susceptibilitywas quantified by determining the number of spores produced on eachgenotype. All the leaves from one plant were collected, their weight wasmeasured, and the leaves were transferred to a 15 ml tube. 500 mldistilled water was added per 100 mg of fresh weight. After vigorousvortexing, spores were counted in a hemocytometer. Mean and standarderror were calculated from 5 repetitions. The experiment was repeated 3times with similar results. Inoculation of LOL1-s lines with thevirulent oomycete pathogen P. parasitica Emco5 was shown to lead toenhanced resistance. Pathogen sporulation on two LOL-s lines was reducedto 5% of wild type and other LOL1-s lines exhibited lesser decreases insusceptibility. Conversely, lol1-as lines showed slight increases insusceptibility. See FIGS. 6A and 6B.

References

The references listed below as well as all references cited in thespecification are incorporated herein by reference to the extent thatthey supplement, explain, provide a background for or teach methodology,techniques and/or compositions employed herein.

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It will be understood that various details of the invention may bechanged without departing from the scope of the invention. Furthermore,the foregoing description is for the purpose of illustration only, andnot for the purpose of limitation the invention being defined by theclaims.

1. An isolated and purified biologically active LOL1 polypeptide thatacts as a positive regulator of programmed cell death in a plant.
 2. Thepolypeptide of claim 1, comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 2 and 4-12, sequences having atleast 85% identity with one of SEQ ID NOs: 2 and 4-12, and fragmentsthereof.
 3. A chimeric polypeptide comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs: 2 and 4-12.
 4. Apolypeptide of claim 1 in a detectably labeled form.
 5. An antibody thatselectively recognizes a polypeptide of claim
 1. 7. An isolated andpurified nucleic acid sequence encoding a polypeptide that acts as apositive regulator of PCD in a plant.
 8. The isolated and purifiednucleic acid sequence of claim 7, wherein the nucleic acid sequencecomprises a nucleic acid sequence selected from the group consisting of:(a) SEQ ID NO: 1; (b) a sequence encoding a polypeptide comprising a anamino acid sequence selected from the group consisting of SEQ ID NOs: 2and 4-12; and (c) a nucleic acid sequence capable of hybridizing understringent conditions to a nucleic acid sequence according to (a) or (b).9. The nucleic acid sequence of claim 7, wherein the nucleic acidsequence is a DNA sequence.
 10. A chimeric gene comprising a nucleicacid sequence of claim 7 or 8 operatively linked to a promoter.
 11. Arecombinant vector comprising the chimeric gene of claim
 10. 12. A hostcell stably transformed with the recombinant vector of claim
 11. 13. Aplant stably transformed with the recombinant vector of claim
 11. 14.The transgenic plant of claim 14, wherein the nucleic acid sequence ispresent in the genome in a copy number effective to confer expression inthe plant of a LOL1 polypeptide that acts as a positive regulator ofprogrammed cell death in a plant.
 15. A seed derived from a transgenicplant of claim
 13. 16. Progeny derived from a transgenic plant of claim13.
 17. A part of a transgenic plant of claim
 13. 18. The transgenicplant of claim 13, wherein the plant is selected from the groupconsisting of Arabidopsis, rice, wheat, barley, rye, corn, potato,carrot, sweet potato, sugar beet, bean, pea, chicory, lettuce, cabbage,cauliflower, broccoli, turnip, radish, spinach, asparagus, onion,garlic, eggplant, pepper, celery, squash, pumpkin, cucumber, apple,pear, quince, melon, plum, cherry, peach, nectarine, apricot,strawberry, grape, raspberry, blackberry, pineapple, avocado, papaya,mango, banana, soybean, tobacco, tomato, sorghum and sugarcane.
 19. Amethod of detecting a nucleic acid molecule that encodes a LOL1polypeptide that acts as a positive regulator of programmed cell deathin a plant in a biological sample containing nucleic acid material, themethod comprising: (a) hybridizing a nucleic acid sequence of claim 7 or8 to nucleic acid material of a biological sample under stringenthybridization conditions, to form a hybridization duplex; and (b)detecting the hybridization duplex.
 20. The method of claim 19, whereinthe detected nucleic acid molecule further comprises a chromosome.
 21. Amethod of identifying positive regulation of programmed cell death in aplant, the method comprising: (a) contacting a query nucleic acidsequence derived from a plant with a probe comprising a nucleic acidsequence of claim 7 or 8; and (b) detecting the formation of ahybridized structure comprising the probe and the query nucleic acidsequence, the presence of a hybridized structure being indicative ofpositive regulation of programmed cell death in the plant.
 22. Themethod of claim 21, wherein the probe comprises a nucleotide sequencecomplementary to a nucleic acid sequence of claim 7 or
 8. 23. The methodof claim 21, wherein the plant is selected from the group consisting ofArabidopsis, rice, wheat, barley, rye, corn, potato, carrot, sweetpotato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower,broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant,pepper, celery, squash, pumpkin, cucumber, apple, pear, quince, melon,plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry,blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco,tomato, sorghum and sugarcane.
 24. A method of increasing LOL1 geneexpression in a plant comprising transforming the plant with therecombinant vector of claim
 11. 25. A method of enhancing diseaseresistance in a plant comprising transforming a plant with therecombinant vector of claim
 11. 26. The method of either of claims 24 or25, wherein the recombinant vector is expressed in the plant at higherlevels than in a wild type plant.
 27. The method of of either of claims24 or 25, wherein the plant is selected from the group consisting ofArabidopsis, rice, wheat, barley, rye, corn, potato, carrot, sweetpotato, sugar beet, bean, pea, chicory, lettuce, cabbage, cauliflower,broccoli, turnip, radish, spinach, asparagus, onion, garlic, eggplant,pepper, celery, squash, pumpkin, cucumber, apple, pear, quince, melon,plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry,blackberry, pineapple, avocado, papaya, mango, banana, soybean, tobacco,tomato, sorghum and sugarcane
 28. An assay kit for detecting thepresence of a LOL1 polypeptide that acts as a positive regulator ofprogrammed cell death in a plant, the assay kit comprising a firstcontainer containing a nucleic acid probe comprising a sequence of tenor more contiguous nucleotide bases corresponding to a fragment of anucleic acid sequence of claim 7 or
 8. 29. The kit of claim 28, furthercomprising a second container containing a detectable moiety.